Tumor-associated peptides binding promiscuosly to human leukocyte antigen (HLA) class II molecules

ABSTRACT

The present invention relates to immunotherapeutic methods, and molecules and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumour-associated T-helper cell peptide epitopes, alone or in combination with other tumour-associated peptides, that serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumour immune responses. In particular, the present invention relates to 49 novel peptide sequences derived from HLA class II molecules of human tumour cell lines which can be used in vaccine compositions for eliciting anti-tumour immune responses.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/912,670, which was filed on Apr. 7, 2008, which is the NationalPhase application of PCT/EP2006/008642, filed Sep. 5, 2006, which claimspriority to European Patent Application No. 05019254.1, filed Sep. 5,2005, the entire contents of which are hereby incorporated.

The present invention relates to immunotherapeutic methods, andmolecules and cells for use in immunotherapeutic methods. In particular,the present invention relates to the immunotherapy of cancer, inparticular renal and colon cancer. The present invention furthermorerelates to tumour-associated T-helper cell peptide epitopes, alone or incombination with other tumour-associated peptides, that serve as activepharmaceutical ingredients of vaccine compositions which stimulateanti-tumour immune responses. In particular, the present inventionrelates to 49 novel peptide sequences derived from HLA class IImolecules of human tumour cell lines which can be used in vaccinecompositions for eliciting anti-tumour immune responses.

For the purposes of the present invention, all references as citedherein are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognised as foreign by the host immune system. The discoveryof the existence of tumour associated antigens has now raised thepossibility of using a host's immune system to intervene in tumourgrowth. Various mechanisms of harnessing both the humoral and cellulararms of the immune system are currently being explored for cancerimmunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognising and destroying tumour cells. The isolation ofcytotoxic T-cells (CTL) from tumour-infiltrating cell populations orfrom peripheral blood suggests that such cells play an important role innatural immune defenses against cancer (Cheever et al., Annals N.Y.Acad. Sci. 1993 690:101-112). CD8-positive T-cells (TCD8⁺) inparticular, which recognise Class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10residues derived from proteins located in the cytosol, play an importantrole in this response. The MHC-molecules in humans are also designatedas human leukocyte-antigens (HLA).

There are two classes of MHC-molecules: MHC-1-molecules andMHC-molecules. MHC-I molecules can be found on most cells having anucleus, and present peptides that result from proteolytic cleavage ofendogenous proteins and larger peptides. MHC-II-molecules can be foundonly on professional antigen presenting cells (APC), and presentpeptides of exogenous proteins that are taken up by APCs during thecourse of endocytosis, and are subsequently processed. Complexes ofpeptide and MHC-I molecules are recognised by CD8-positive cytotoxicT-lymphocytes, and complexes of peptide and MHC-II molecules arerecognised by CD4-positive-helper-T-cells.

CD4-positive helper T-cells play an important role in orchestrating theeffector functions of anti-tumor T-cell responses and for this reasonthe identification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) may be of great importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Kobayashi, H., R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J. J. Lasarte,M. Herraiz, B. Sangro, J. Prieto, F. Borras-Cuesta, and E. Celis. 2002.Identification of an antigenic epitope for helper T lymphocytes fromcarcinoembryonic antigen. Clin. Cancer Res. 8:3219-3225, Gnjatic, S., D.Atanackovic, E. Jäger, M. Matsuo, A. Selvakumar, N. K. Altorki, R. G.Maki, B. Dupont, G. Ritter, Y. T. Chen, A. Knuth, and L. J. Old. 2003.Survey of naturally occurring CD4+ T-cell responses against NY-ESO-1 incancer patients: Correlation with antibody responses. Proc. Natl. Acad.Sci. U.S.A. 100(15):8862-7).

It was shown in mammalian animal models, e.g., mice, that even in theabsence of cytotoxic T lymphocyte (CTL) effector cells (i.e.,CD8-positive T lymphocytes), CD4-positive T-cells are sufficient forinhibiting visualization of tumors via inhibition of angiogenesis bysecretion of interferon-gamma (IFN) (Qin, Z. and T. Blankenstein. 2000.CD4+ T-cell-mediated tumor rejection involves inhibition of angiogenesisthat is dependent on IFN gamma receptor expression by nonhematopoieticcells. Immunity. 12:677-686). Additionally, it was shown thatCD4-positive T-cells recognizing peptides from tumor-associated antigenspresented by HLA class II molecules can counteract tumor progression viathe induction of an Antibody (Ab) responses (Kennedy, R. C., M. H.Shearer, A. M. Watts, and R. K. Bright. 2003. CD4⁺ T lymphocytes play acritical role in antibody production and tumor immunity against simianvirus 40 large tumor antigen. Cancer Res. 63:1040-1045). In contrast totumor-associated peptides binding to HLA class I molecules, only a smallnumber of class II ligands of TAA have been described so far(www.cancerimmunity.org, www.syfpeithi.de). Since the constitutiveexpression of HLA class II molecules is usually limited to cells of theimmune system (Mach, B., V. Steimle, E. Martinez-Soria, and W. Reith.1996. Regulation of MHC class II genes: lessons from a disease. Annu.Rev. Immunol. 14:301-331), the possibility of isolating class IIpeptides directly from primary tumors was not considered possible.Therefore, numerous strategies to target antigens into the class IIprocessing pathway of antigen presenting cells (APCs) have beendescribed. For example, the APCs having been incubated with the antigenof interest to enable it to be taken up, processed and presented (Chaux,P., V. Vantomme, V. Stroobant, K. Thielemans, J. Corthals, R. Luiten, A.M. Eggermont, T. Boon, and B. P. van der Bruggen. 1999. Identificationof MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) Tlymphocytes. J. Exp. Med. 189:767-778), or cells have been transfectedwith genes or minigenes encoding the antigen of interest and fused tothe invariant chain, which mediates the translocation of antigens to thelysosomal MHC class II processing and assembling compartment (MIIC).

For a peptide to trigger (elicit) a cellular immune response, it mustbind to an MHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-1-binding peptides are usually 8-10 residues inlength and contain two conserved residues (“anchor”) in their sequencethat interact with the corresponding binding groove of the MHC-molecule.

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells.

The antigens that are recognised by the tumour specific cytotoxicT-lymphocytes, that is, their epitopes, can be molecules derived fromall protein classes, such as enzymes, receptors, transcription factors,etc. Furthermore, tumour associated antigens, for example, can also bepresent in tumour cells only, for example as products of mutated genesor from alternative open reading frames (ORFs), or from protein splicing(Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, vander Bruggen P, Boon T, Van den Eynde B J. An antigenic peptide producedby peptide splicing in the proteasome. Science. 2004 Apr. 23; 304(5670):587-90). Another important class of tumour associated antigensare tissue-specific structures, such as CT (“cancer testis”)-antigensthat are expressed in different kinds of tumours and in healthy tissueof the testis.

Various tumour associated antigens have been identified. Further, muchresearch effort is being expended to identify additional tumourassociated antigens. Some groups of tumour associated antigens, alsoreferred to in the art as tumour specific antigens, are tissue specific.Examples include, but are not limited to, tyrosinase for melanoma, PSAand PSMA for prostate cancer and chromosomal cross-overs such as bcr/ablin lymphoma. However, many tumour associated antigens that have beenidentified occur in multiple tumour types, and some, such as oncogenicproteins and/or tumour suppressor genes (tumour suppressor genes are,for example reviewed for renal cancer in Linehan W M, Walther M M, ZbarB. The genetic basis of cancer of the kidney. J. Urol. 2003 December;170(6 Pt 1):2163-72) which actually cause the transformation event,occur in nearly all tumour types. For example, normal cellular proteinsthat control cell growth and differentiation, such as p53 (which is anexample for a tumour suppressor gene), ras, c-met, myc, pRB, VHL, andHER-2/neu, can accumulate mutations resulting in upregulation ofexpression of these gene products thereby making them oncogenic(McCartey et al. Cancer Research 1998 15:58 2601-5; Disis et al. CibaFound. Symp. 1994 187:198-211). These mutant proteins can be the targetof a tumour specific immune response in multiple types of cancer.

For the proteins to be recognised by the cytotoxic T-lymphocytes astumour-specific antigen, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumour cells and not by normal healthy tissues or at the very least,expressed in rather small amounts in normal healthy tissue. It isfurthermore desirable that the respective antigen is not only present inone type of tumour, but also in high concentrations (e.g. copy numbersper cell). The presence of epitopes in the amino acid sequence of theantigen is essential, since such peptide (“immunogenic peptide”) that isderived from a tumour associated antigen should lead to an in vitro orin vivo T-cell-response.

Until now, numerous strategies to target antigens into the class IIprocessing pathway have been described. It is possible to incubateantigen presenting cells (APCs) with the antigen of interest to be takenup and processed (Chaux, P., Vantomme, V., Stroobant, V., Thielemans,K., Corthals, J., Luiten, R., Eggermont, A. M., Boon, T. & van der, B.P. (1999) J. Exp. Med. 189, 767-778). Other strategies use fusionproteins that contain lysosomal target sequences. Expressed in APCs,such fusion proteins direct the antigens into the class II processingcompartment (Marks, M. S., Roche, P. A., van Donselaar, E., Woodruff,L., Peters, P. J. & Bonifacino, J. S. (1995) J. Cell Biol. 131, 351-369,Rodriguez, F., Harkins, S., Redwine, J. M., de Pereda, J. M. & Whitton,J. L. (2001) J. Virol. 75, 10421-10430).

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumour immunity. T-helper cell epitopes thattrigger a T-helper cell response of the TH1 type support effectorfunctions of CD8-positive Killer T-cells, which include cytotoxicfunctions directed against tumour cells displaying tumour-associatedpeptide/MHC complexes on their cell surfaces. In this waytumour-associated T-helper cell peptide epitopes, alone or incombination with other tumour-associated peptides, can serve as activepharmaceutical ingredients of vaccine compositions which stimulateanti-tumour immune responses.

The major task in the development of a tumour vaccine is therefore theidentification and characterisation of novel tumour associated antigensand immunogenic T-helper epitopes derived therefrom, that can berecognised by CD4-positive CTLs. Therefore, there is a need to providenovel amino acid sequences for peptides that have the ability to bind toa molecule of the human major histocompatibility complex (MHC) class-II.The present invention fulfils this need.

SUMMARY OF THE INVENTION

According to the present invention, a tumour associated peptide that isselected from the group of peptides comprising at least one sequenceaccording to any of SEQ ID NO: 1 to SEQ ID NO: 49 is provided, whereinthe peptide has the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-II, provided that the peptide isnot the intact human tumour associated polypeptide.

In another embodiment, tumor associated peptides of the presentinvention consist essentially of an amino acid sequence according to anyof SEQ ID NO: 1 to SEQ ID NO: 49. Preferably the tumor associatedpeptide exhibits an overall length of between 9 and 100, and morepreferably between 9 and 30 amino acids.

More preferably, tumor associated peptides of the present inventionconsist of an amino acid sequence according to any of SEQ ID NO: 1 toSEQ ID NO: 49. Preferably, tumour associated peptides of the presentinvention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-II, in particular toHLA-DRB1*0101.

In another embodiment, tumor associated peptides of the presentinvention have the ability to bind to at least one additional moleculeof the human major histocompatibility complex (MHC) class-II.

Tumor associated peptides of the present invention may includenon-peptide bonds.

In another embodiment, tumor associated peptides of the presentinvention may comprise a fusion protein comprising N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (Ii).

The present invention also provides a nucleic acid, encoding a tumorassociated peptide of the invention. The nucleic acid may be DNA, cDNA,PNA, CNA, RNA or combinations thereof.

Another embodiment of the present invention provides expression vectorscapable of expressing a nucleic acid encoding a tumor associated peptideof the invention.

The present invention further provides a host cell comprising a nucleicacid according r an expression vector of the invention. Preferably, thehost cell is a recombinant RCC or A wells cell.

In another embodiment of the invention, there is provided a method ofproducing a tumor associated peptide of the invention by culturing ahost cell described above and isolating the peptide from the host cellor its culture medium.

The present invention also provides a pharmaceutical compositioncomprising a tumor associated peptide, a nucleic acid or an expressionvector as described above and a pharmaceutically acceptable carrier. Thepharmaceutical composition may be in the form of a cancer vaccine, andmay optionally comprise at least one suitable adjuvant. The presentinvention further provides use of tumor associated peptides, nucleicacids expression vectors of the invention in medicine.

Another embodiment of the present invention provides methods of killingtarget cells in a patient, which target cells aberrantly express apolypeptide comprising an amino acid sequence disclosed in SEQ IDNO:1-49. The methods comprise administering to the patient an effectiveamount of a tumor associated peptide, a nucleic acid, or an expressionvector of the present invention, wherein the amount of the peptide,nucleic acid or expression vector is effective to provoke an anti-targetcell immune response in the patient.

The present invention also provides for the use of a tumour associatedpeptide, a nucleic acid, or an expression vector of the invention in themanufacture of a medicament for killing target cells in a patient, whichtarget cells aberrantly express a polypeptide comprising an amino acidsequence disclosed in SEQ ID NO:1-49.

The medicament may be used for inducing an immune response, inparticular a cellular immune response, more particularly a T-cellmediated immune response against cells of solid tumors, which cellsexpress a human class II MHC molecule on their surface and present apolypeptide comprising an amino acid sequence disclosed in SEQ IDNO:1-49.

The present invention further provides an in vitro method for producingactivated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell for aperiod of time sufficient to activate said CTL in an antigen specificmanner, wherein the antigen is a tumor associated peptide of the presentinvention. In certain embodiments the antigen is loaded onto class IIMHC molecules expressed on the surface of a suitable antigen-presentingcell by contacting a sufficient amount of the antigen with anantigen-presenting cell. In certain embodiments the antigen-presentingcell comprises an expression vector of the present invention. In certainpreferred embodiments, the class II MHC molecule is HLA-DRB1*0101.

Another embodiment of the present invention provides activated cytotoxicT lymphocytes (CTL), produced by the method described above and whichselectively recognize a cell that aberrantly expresses a polypeptidecomprising an amino acid sequence presented in SEQ ID NO:1-49.

The present invention also provides a T-cell receptor (TCR), whichrecognizes a cell that aberrantly expresses a polypeptide comprising anamino acid sequence given SEQ ID NO: 1-49, the TCR being obtainable fromthe cytotoxic T lymphocyte (CTL) of described above, or a functionallyequivalent molecule to the TCR. The present invention provides nucleicacids encoding T-cell receptors (TCR) of the present invention and alsoprovides expression vectors capable of expressing T-cell receptors (TCR)of the present invention.

Another embodiment of the present invention provides a method of killingtarget cells in a patient, which target cells aberrantly express apolypeptide comprising an amino acid sequence presented in SEQ IDNO:1-49. The method comprises administering to the patient an effectivenumber of cytotoxic T lymphocytes (CTL) of the present invention and asdescribed above.

The present invention further provides a method of killing target cellsin a patient, which target cells aberrantly express a polypeptidecomprising an amino acid sequence as disclosed in SEQ ID NO:1-49. Themethod comprises the steps of: (1) obtaining cytotoxic T lymphocytes(CTL) from the patient; (2) introducing into the cells a nucleic acidencoding a T-cell receptor (TCR) of the present invention, or afunctionally equivalent molecule; and (3) introducing the cells producedin step (2) into the patient.

In certain preferred embodiments, the target cells are cancer cells, inparticular cells of solid tumor that express a human class II MHCmolecule on their surface and present a polypeptide comprising an aminoacid sequence as presented in SEQ ID NO:1-49.

Another embodiment of the present invention provides the use ofcytotoxic T lymphocytes of the present invention in the manufacture of amedicament for killing target cells in a patient, which target cellsaberrantly express a polypeptide comprising an amino acid sequence givenin SEQ ID NO:1-49.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the expression of HLA class II molecules in RCC of threepatients. Whereas in the tumor of patient RCC132, the HLA positive cellswere preferably localized at the margin (A,B) the HLA class IIexpression patterns of the tumors from patient RCC190 and RCC211revealing a more papillary structure were more evenly spread (C,E,G).The visualization of CD68-positive macrophages (B,D,F) in serial tissuesections illustrates a close spatial relationship of tumor-infiltratingmononuclear immune cells and HLA II expressing tumor cells. Incubationwith mouse IgG instead of specific antibodies consistently revealednegative staining results (H). Capital T marks the tumor.

FIG. 2 shows a FACS analysis of CD4-positive T-cells specific forIGFBP3₁₆₉₋₁₈₁, MMP7₂₄₇₋₂₆₂ and CCND1₁₉₈₋₂₁₂. Shown are representativedot blots of intracellular IFNγ staining against CD4-FITC.

FIG. 3 shows a schematic illustration of antigen-specific IFNγ producingCD4-positive T-cells detected in each donor and for each peptide. Shownis the percentage of IFNγ producing CD4-positive T-cells for each donorand peptide used for stimulation. Cells were incubated in 96-wellplates—7 wells per donor and per peptide. Boxed are values considered aspositive: percentage of IFNγ producing CD4-positive T-cells was morethan two-fold higher compared with negative control without peptide.Percentages of IFNγ producing CD4-positive T-cells detected afterstimulation with irrelevant peptide correlated with values afterstimulation without peptide, with the exception of Donor 1 after the3^(rd) stimulation with IGFBP3₁₆₉₋₁₈₁. However, this effect was not seenanymore after the 4^(th) stimulation.

FIG. 4 shows the expression of HLA class II molecules in CCA165(moderately differentiated adenocarcinoma of the colon). In the laminapropria of areas with normal colonic mucosa (panel c and left side ofpanel a, marked by asterisk) typically some HLA class II positivemacrophages are observed but epithelial cells were consistently negativefor HLA class II expression. In epithelial cells from different areas ofthe tumor, however, a pronounced expression of HLA II was noted as shownon the right side of panel a, and in panel b and d.

FIGS. 5 a and 5 a show the identification of peptide sequence ofpeptides eluted from HLA-Class II molecules isolated from primary humantumor tissue by mass spectroscopy. FIG. 5 a: fragments derived fromfragmentation of naturally processed and presented HLA Class II ligandfrom MMP7 corresponding to the peptide sequence with SEQ ID NO: 1(SQDDIKGIQKLYGKRS). Annotated fragments are depicted in Table 5. FIG. 5b: fragments derived from fragmentation of synthetic peptide having thepeptide sequence of SEQ ID NO: 1. Fragmentations of both synthetic andnaturally processed peptides yield equivalent fragmentation patterns andallow deduction and confirmation of the primary amino acid sequence ofthe previously uncharacterized peptide sequence (SEQ ID NO: 1) of thisHLA class II ligand from human MMP7.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the inventors demonstrate that it is possibleto isolate and characterize peptides binding to HLA class II moleculesdirectly from mammalian tumors, preferentially human tumors,preferentially solid tumors, e.g., from renal cell carcinomas and coloncarcinomas. Infiltrating monocytes expressed MHC class II molecules aswell as tumor cells, and, in addition, tumor cells showed up-regulationof several cytokine or chemokine-induced gene products, e.g., interferongamma-induced gene products.

The present invention provides peptides stemming from antigensassociated with tumorigenesis, and the ability to bind sufficiently toHLA class II molecules for triggering an immune response of humanleukocytes, especially lymphocytes, especially T lymphocytes, especiallyCD4-positive T lymphocytes, especially CD4-positive T lymphocytesmediating T_(H1)-type immune responses. The peptides stem fromtumor-associated antigens, especially tumor-associated antigens withfunctions in, e.g., proteolysis, angiogenesis, cell growth, cell cycleregulation, cell division, regulation of transcription, regulation oftranslation, tissue invasion, including, e.g., tumor-associated peptidesfrom matrix-metalloproteinase 7 (MMP7; SEQ ID NO: 1) and insulin-likegrowth factor binding protein 3 (IGFBP3; SEQ ID NO: 25).

In the present invention the inventors also provide conclusive evidencethat tumor-associated peptides sufficiently bind promiscuously toHLA-class II molecules, especially those HLA class II allelesgenetically encoded by HLA DR loci of the human genome, are able toelicit immune responses mediated by human CD4-positive T-cells.CD4-positive T-cells were isolated from human peripheral blood,demonstrating that the claimed peptides are suitable for triggeringT-cell responses of the human immune system against selected peptides ofthe tumor cell peptidome. As peptides can be synthesized chemically andcan be used as active pharmaceutical ingredients of pharmaceuticalpreparations, the peptides provided by the inventors' invention can beused for immunotherapy, preferentially cancer immunotherapy.

To identify HLA class II ligands from TAA for the development ofpeptide-based immunotherapy, the inventors attempted to isolateHLA-DR-presented peptides directly from dissected solid tumors, inparticular from renal cell carcinoma (RCC), which had been reported tobe able to express class II molecules (Gastl, G., T. Ebert, C. L.Finstad, J. Sheinfeld, A. Gomahr, W. Aulitzky, and N. H. Bander. 1996.Major histocompatibility complex class I and class II expression inrenal cell carcinoma and modulation by interferon gamma. J. Urol.155:361-367). Even if the majority of tumor cells were class IInegative, state-of-the-art mass spectrometers should provide thesensitivity required for identification of class II peptides fromminimal numbers of tumor cells, or from infiltrating leukocytes whichmight cross-present TAA, or from stromal cells in the perimeter of thegrowing tumor.

The reasons for focusing on RCC to demonstrate technical proof ofconcept were the following: Around 150,000 people worldwide are newlydiagnosed with RCC each year, the disease is associated with a highmortality rate, which results in approximately 78,000 deaths per annum(Pavlovich, C. P. and L. S. Schmidt. 2004. Searching for the hereditarycauses of renal-cell carcinoma. Nat. Rev. Cancer 4:381-393). Ifmetastases are diagnosed, the one-year survival rate decreases toapproximately 60% (Jemal, A., R. C. Tiwari, T. Murray, A. Ghafoor, A.Samuels, E. Ward, E. J. Feuer, and M. J. Thun. 2004. Cancer statistics,2004. CA Cancer J. Clin. 54:8-29), underlining the high unmet medicalneed in this indication. Because RCC seems to be an immunogenic tumor(Oliver R T D, Mehta A, Barnett M J. A phase 2 study of surveillance inpatients with metastatic renal cell carcinoma and assessment of responseof such patients to therapy on progression. Mol. Biother. 1988; 1:14-20.Gleave M, Elhilali M, Frodet Y, et al. Interferon gamma-1b compared withplacebo in metastatic renal cell carcinoma. N Engl J. Med. 1998;338:1265), as indicated by the existence of tumor-reacting andtumor-infiltrating CTL (Finke, J. H., P. Rayman, J. Alexander, M.Edinger, R. R. Tubbs, R. Connelly, E. Pontes, and R. Bukowski. 1990.Characterization of the cytolytic activity of CD4-positive andCD8-positive tumor-infiltrating lymphocytes in human renal cellcarcinoma. Cancer Res. 50:2363-2370), clinical trials have beeninitiated to develop peptide-based anti-tumor vaccinations (Wierecky J,Mueller M, Brossart P. Dendritic cell-based cancer immunotherapytargeting MUC-1. Cancer Immunol Immunother. 2005 Apr. 28). However, dueto the lack of helper T-cell epitopes from TAA, molecularly definedvaccines usually comprise peptides functioning as class I ligands only.

In the scientific work leading to the present invention, the inventorswere able to isolate class II ligands from ten RCC samples, threecolorectal carcinomas (CCA) and one transitional cell carcinoma (TCC,urothelial carcinoma). Only selected of the ligands from TAA identifiedby this approach have the unifying capacity to

-   -   1. Stem from antigens with known tumor association;    -   2. Bind to the most common HLA class II DR allele, HLA        DRB1*0101; and    -   3. Have characteristics setting them apart from the majority of        HLA class II ligands, in that they fulfill criteria regarding        their primary amino acid sequence allowing them to promiscuously        bind to HLA-DR molecules from at least two different alleles.

As exemplified below with a peptide from MMP7 (SEQ ID NO: 1), thesepromiscuously HLA-DR-binding, tumor-associated peptides were found to berecognized by CD4-positive T-cells.

A first aspect of the invention provides a peptide, comprising an aminoacid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 49 or avariant thereof provided that the peptide is not the intact humanpolypeptide from which the amino acid sequence is derived (i.e. one ofthe full-length sequences as listed in the locus link IDs (Accessionnumbers, see the attached Table 1, below).

As described herein below, the peptides that form the basis of thepresent invention have all been identified as being presented by MHCclass II bearing cells (RCC). Thus, these particular peptides as well asother peptides containing the sequence (i.e. derived peptides) will mostlikely all elicit a specific T-cell response, although the extent towhich such response will be induced might vary from individual peptideto peptide. Differences, for example, could be caused due to mutationsin said peptides (see below). The person of skill in the present art iswell aware of methods that can be applied in order to determine theextent to which a response is induced by an individual peptide, inparticular with reference to the examples herein and the respectiveliterature.

Preferably, a peptide according to the present invention consistsessentially of an amino acid sequence according to any of SEQ ID NO: 1to SEQ ID NO: 49 or a variant thereof.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 49 or a variant thereof, contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as core sequenceof the peptide comprising the binding motif and as an immunogenicT-helper epitope.

Nevertheless, these stretches can be important in order to provide foran efficient introduction of the peptide according to the presentinvention into the cells. In one embodiment of the present invention,the peptide of the present invention comprises the 80 N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (p33, in thefollowing “Ii”) as derived from the NCBI, GenBank Accession-numberX00497 (Strubin, M., Mach, B. and Long, E. O. The complete sequence ofthe mRNA for the HLA-DR-associated invariant chain reveals a polypeptidewith an unusual transmembrane polarity EMBO J. 3 (4), 869-872 (1984)).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence. For example, a peptide may be modified so that it atleast maintains, if not improves, the ability to interact with and binda suitable MHC molecule, such as HLA-A, and so that it at leastmaintains, if not improves, the ability to generate activated CTL whichcan recognize and kill cells which express a polypeptide which containsan amino acid sequence as defined in the aspects of the invention. Ascan derived from the database as described in the following, certainpositions of HLA-A binding peptides are typically anchor residuesforming a core sequence fitting to the binding motif of the HLA bindinggroove.

Those amino acid residues that are not essential to interact with the Tcell receptor can be modified by replacement with another amino acidwhose incorporation does not substantially effect T cell reactivity anddoes not eliminate binding to the relevant MHC. Thus, apart from theproviso given, the peptide of the invention may be any peptide (by whichterm the inventors include oligopeptide or polypeptide) which includesthe amino acid sequences or a portion or variant thereof as given.

It is furthermore known for MHC-class II presented peptides that thesepeptides are composed of a “core sequence” having a certain HLA-specificamino acid motif and, optionally, N- and/or C-terminal extensions thatdo not interfere with the function of the core sequence (i.e. are deemedas irrelevant for the interaction of the peptide and the T-cell). The N-and/or C-terminal extensions can be between 1 to 10 amino acids inlength, respectively. Thus, a preferred peptide of the present inventionexhibits an overall length of between 9 and 100, preferably between 9and 30 amino acids. These peptide can be used either directly in orderto load MHC class II molecules or the sequence can be cloned into thevectors according to the description herein below. As these peptidesform the final product of the processing of larger peptides within thecell, longer peptides can be used as well. The peptides of the inventionmay be of any size, but typically they may be less than 100,000 inmolecular weight, preferably less than 50,000, more preferably less than10,000 and typically about 5,000. In terms of the number of amino acidresidues, the peptides of the invention may have fewer than 1000residues, preferably fewer than 500 residues, more preferably fewer than100 residues.

If a peptide that is greater than around 12 amino acid residues is useddirectly to bind to a MHC molecule, it is preferred that the residuesthat flank the core HLA binding region are ones that do notsubstantially affect the ability of the peptide to bind specifically tothe binding groove of the MHC molecule or to present the peptide to theCTL. However, as already indicated above, it will be appreciated thatlarger peptides may be used, especially when encoded by apolynucleotide, since these larger peptides may be fragmented bysuitable antigen-presenting cells.

Examples for peptides of MHC ligands, motifs, variants, as well ascertain examples for N- and/or C-terminal extensions can be, forexample, derived from the database SYFPEITHI (Rammensee H, Bachmann J,Emmerich N P, Bachor O A, Stevanovic S. SYFPEITHI: database for MHCligands and peptide motifs. Immunogenetics. 1999 November; 50(3-4):213-9at http://syfpeithi.bmi-heidelberg.com/ and the references as citedtherein.

As non-limiting examples, certain peptides for HLA-DR in the databaseare K H K V Y A C E V T H Q G L S S derived from Ig kappa chain 188-203(Kovats et al. Eur J. Immunol. 1997 April; 27(4):1014-21); K V Q W K V DN A L Q S G N S derived from Ig kappa chain 145-159 (Kovats et al. EurJ. Immunol. 1997 April; 27(4):1014-21), L P R L I A F T S E H S H Fderived from GAD65 270-283 (Endl et al. J Clin Invest. 1997 May 15;99(10):2405-15) or F F R M V I S N P A A T H Q D I D F L I derived fromGAD65 556-575 (Endl et al. J Clin Invest. 1997 May 15; 99(10):2405-15).In addition, peptides can also be derived from mutated sequences ofantigens, such as in the case of A T G F K Q S S K A L Q R P V A Sderived from bcr-abl 210 kD fusion protein (ten Bosch et al. Blood. 1996Nov. 1; 88(9):3522-7), G Y K V L V L N P S V A A T derived from HCV-1NS3 28-41 Diepolder et al. J. Virol. 1997 August; 71(8):6011-9), or F RK Q N P D I V I Q Y M D D L Y V G derived from HIV-1 (HXB2) RT 326-345(van der Burg et al. J. Immunol. 1999 Jan. 1; 162(1):152-60). All“anchor” amino acids (see Friede et al., Biochim Biophys Acta. 1996 Jun.7; 1316(2):85-101; Sette et al. J. Immunol. 1993 Sep. 15;151(6):3163-70; Hammer et al. Cell. 1993 Jul. 16; 74(1):197-203., andHammer et al. J Exp Med. 1995 May 1; 181(5):1847-55. As examples forHLA-DR4) have been indicated in bold, the putative core sequences havebeen underlined.

All the above described peptides are encompassed by the term “variants”of the given amino acid sequence.

By “peptide” the inventors include not only molecules in which aminoacid residues are joined by peptide (—CO—NH—) linkages but alsomolecules in which the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that, at leastfor MHC class II and T helper cell responses, these pseudopeptides areuseful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

Typically, the peptide of the invention is one which, if expressed in anantigen presenting cell, may be processed so that a fragment is producedwhich is able to bind to an appropriate MHC molecule and may bepresented by a suitable cell and elicit a suitable T-cell response. Itwill be appreciated that a fragment produced from the peptide may alsobe a peptide of the invention. Conveniently, the peptide of theinvention contains a portion that includes the given amino acid sequenceor a portion or variant thereof and a further portion which confers somedesirable property. For example, the further portion may include afurther T-cell epitope (whether or not derived from the same polypeptideas the first T-cell epitope-containing portion) or it may include acarrier protein or peptide. Thus, in one embodiment the peptide of theinvention is a truncated human protein or a fusion protein of a proteinfragment and another polypeptide portion provided that the human portionincludes one or more inventive amino acid sequences.

In a particularly preferred embodiment, the peptide of the inventionincludes the amino acid sequence of the invention and at least onefurther T-cell epitope wherein the further T-cell epitope is able tofacilitate the production of a T-cell response directed at the type oftumour that aberrantly expresses a tumour-associated antigen. Thus, thepeptides of the invention include so-called “beads on a string”polypeptides which can also be used as vaccines.

It will be appreciated from the following that in some applications thepeptides of the invention may be used directly (i.e. they are notproduced by expression of a polynucleotide in a patient's cell or in acell given to a patient); in such applications it is preferred that thepeptide has fewer than 100 or 50 residues. A preferred peptide of thepresent invention exhibits an overall length of between 9 and 30 aminoacids.

Preferably, the peptides of the invention are able to bind to HLA-DR.More preferably, the peptides bind selectively to HLA-DRB1*0101.

In another aspect of the present invention, similar to the situation asexplained above for MHC class II molecules, the peptides of theinvention may be used to trigger an MHC class I specific T cellresponse. A preferred MHC class I specific peptide of the presentinvention exhibits an overall length of between 9 and 16, preferablybetween 9 and 12 amino acids. It shall be understood that those peptidesmight be used (for example in a vaccine) as longer peptides, similar toMHC class II peptides. Methods to identify MHC class I specific “Coresequences” having a certain HLA-specific amino acid motif for HLA classI-molecules are known to the person of skill and can be predicted, forexample, by the computer programs PAProC and SYFPEITHI.

The peptides of the invention are particularly useful inimmunotherapeutic methods to target and kill cells that aberrantlyexpress polypeptides that form the basis for the present peptides of theinvention. Since these specific peptides consisting of the given aminoacid sequences bind to HLA-DR it is preferred that the peptides of theinvention are ones that bind HLA-DR and when so bound, theHLA-DR-peptide complex when present on the surface of a suitableantigen-presenting cell, is capable of eliciting the production of a CTLthat recognises a cell that aberrantly expresses a polypeptidecomprising the given amino acid sequence.

In one embodiment of the present invention, the peptide of the presentinvention comprises the 80 N-terminal amino acids of the HLA-DRantigen-associated invariant chain (p33, in the following “Ii”) asderived from the NCBI, GenBank Accession-number X00497 (see also below).

By “aberrantly expressed” we include the meaning that the polypeptide isover-expressed compared to normal levels of expression or that the geneis silent in the tissue from which the tumour is derived but in thetumour it is expressed. By “over-expressed” we mean that the polypeptideis present at a level at least 1.2× that present in normal tissue;preferably at least 2× and more preferably at least 5× or 10× the levelpresent in normal tissue.

Peptides (at least those containing peptide linkages between amino acidresidues) may be synthesised by the Fmoc-polyamide mode of solid-phasepeptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is effected using 20%piperidine in N,N-dimethylformamide. Side-chain functionalities may beprotected as their butyl ethers (in the case of serine threonine andtyrosine), butyl esters (in the case of glutamic acid and asparticacid), butyloxycarbonyl derivative (in the case of lysine andhistidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalisingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/lhydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethandithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesized. Also a combination of solid phase and solution phasemethodologies for the synthesis of peptides is possible (see, forexample, Bruckdorfer T, Marder O, Albericio F. From production ofpeptides in milligram amounts for research to multi-tons quantities fordrugs of the future. Curr Pharm Biotechnol. 2004 February; 5(1):29-43and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilisation of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available fromCalbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be effected by any one, or a combination of, techniquessuch as size exclusion chromatography, ion-exchange chromatography,hydrophobic interaction chromatography and (usually) reverse-phase highperformance liquid chromatography using acetonitril/water gradientseparation.

Analysis of peptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a peptide of the invention. The polynucleotidemay be DNA, cDNA, PNA, CNA, RNA or combinations thereof and it may ormay not contain introns so long as it codes for the peptide. Of course,it is only peptides that contain naturally occurring amino acid residuesjoined by naturally occurring peptide bonds that are encodable by apolynucleotide. A still further aspect of the invention provides anexpression vector capable of expressing a polypeptide according to theinvention.

A variety of methods have been developed to operably linkpolynucleotides, especially DNA, to vectors for example viacomplementary cohesive termini. For instance, complementary homopolymertracts can be added to the DNA segment to be inserted to the vector DNA.The vector and DNA segment are then joined by hydrogen bonding betweenthe complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3′-single-stranded terminiwith their 3′-5′-exonucleolytic activities, and fill in recessed 3′-endswith their polymerising activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyse the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA encoding the polypeptide of theinvention is to use the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487-491. This method may be used forintroducing the DNA into a suitable vector, for example by engineeringin suitable restriction sites, or it may be used to modify the DNA inother useful ways as is known in the art. In this method the DNA to beenzymatically amplified is flanked by two specific primers whichthemselves become incorporated into the amplified DNA. The said specificprimers may contain restriction endonuclease recognition sites which canbe used for cloning into expression vectors using methods known in theart.

The DNA (or in the case of retroviral vectors, RNA) is then expressed ina suitable host to produce a polypeptide comprising the compound of theinvention. Thus, the DNA encoding the polypeptide constituting thecompound of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed in U.S. Pat. Nos. 4,440,859 issued 3 Apr. 1984 to Rutter etal.; 4,530,901 issued 23 Jul. 1985 to Weissman; 4,582,800 issued 15 Apr.1986 to Crowl; 4,677,063 issued 30 Jun. 1987 to Mark et al.; 4,678,751issued 7 Jul. 1987 to Goeddel; 4,704,362 issued 3 Nov. 1987 to Itakuraet al.; 4,710,463 issued 1 Dec. 1987 to Murray; 4,757,006 issued 12 Jul.1988 to Toole, Jr. et al.; 4,766,075 issued 23 Aug. 1988 to Goeddel etal.; and 4,810,648 issued 7 Mar. 1989 to Stalker, all of which areincorporated herein by reference.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells. Preferably, the system can be RCC orAwells cells.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention. Typicalprokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99Aand pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells. An example of an inducible mammalian expression vector is pMSG,also available from Pharmacia. This vector uses theglucocorticoid-inducible promoter of the mouse mammary tumour virus longterminal repeat to drive expression of the cloned gene. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).Other vectors and expression systems are well known in the art for usewith a variety of host cells.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and kidneycell lines. Yeast host cells include YPH499, YPH500 and YPH501 which aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Preferred mammalian host cells include Chinese hamster ovary(CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryocells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derivedCOS-1 cells available from the ATCC as CRL 1650 and 293 cells which arehuman embryonic kidney cells. Preferred insect cells are Sf9 cells whichcan be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (1972) Proc.Natl. Acad. Sci. USA 69,2110 and Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Transformation of yeast cells is described in Sherman et al(1986) Methods In Yeast Genetics, A Laboratory Manual, Cold SpringHarbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is alsouseful. With regard to vertebrate cells, reagents useful in transfectingsuch cells, for example calcium phosphate and DEAE-dextran or liposomeformulations, are available from Stratagene Cloning Systems, or LifeTechnologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is alsouseful for transforming and/or transfecting cells and is well known inthe art for transforming yeast cell, bacterial cells, insect cells andvertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an expressionconstruct of the present invention can be grown to produce thepolypeptide of the invention. Cells can be harvested and lysed and theirDNA content examined for the presence of the DNA using a method such asthat described by Southern (1975) J. Mol. Biol. 98,503 or Berent et al.;(1985) Biotech. 3,208. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well known immunologicalmethods when the recombinant DNA is capable of directing the expressionof the protein. For example, cells successfully transformed with anexpression vector produce proteins displaying appropriate antigenicity.Samples of cells suspected of being transformed are harvested andassayed for the protein using suitable antibodies. Thus, in addition tothe transformed host cells themselves, the present invention alsocontemplates a culture of those cells, preferably a monoclonal (clonallyhomogeneous) culture, or a culture derived from a monoclonal culture, ina nutrient medium.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules.

A further aspect of the invention provides a method of producing apeptide for intravenous (i.v.) injection, sub-cutaneous (s.c.)injection, intradermal (i.d.) injection, intraperitoneal (i.p.)injection, intramuscular (i.m.) injection. Preferred methods of peptideinjection are s.c., i.d., i.p., i.m., and i.v. Preferred ways of DNAinjection are i.d., i.m., s.c., i.p. and i.v. Doses of between 1 and 500mg of peptide or DNA may be given.

A further aspect of the invention relates to the use of a tumourassociated peptide according to the invention, a nucleic acid accordingto the invention or an expression vector according to the invention inmedicine.

A further aspect of the invention provides a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising an amino acid sequence of the invention, the methodcomprising administering to the patient an effective amount of a peptideaccording to the invention, or an effective amount of a polynucleotideor an expression vector encoding a said peptide, wherein the amount ofsaid peptide or amount of said polynucleotide or expression vector iseffective to provoke an anti-target cell immune response in saidpatient. The target cell is typically a tumour or cancer cell.

The peptide or peptide-encoding nucleic acid constitutes a tumour orcancer vaccine. It may be administered directly into the patient, intothe affected organ or systemically, or applied ex vivo to cells derivedfrom the patient or a human cell line which are subsequentlyadministered to the patient, or used in vitro to select a subpopulationfrom immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant such as Detox, or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker et al (1993) Ann. NYAcad. Sci. 690, 276-291). The peptide may also be tagged, or be a fusionprotein, or be a hybrid molecule. The peptides whose sequence is givenin the present invention are expected to stimulate CD4 CTL. However,stimulation is more efficient in the presence of help provided byCD4-positive T-cells. Thus, the fusion partner or sections of a hybridmolecule suitably provide epitopes which stimulate CD4-positive T-cells.CD4-positive stimulating epitopes are well known in the art and includethose identified in tetanus toxoid. The polynucleotide may besubstantially pure, or contained in a suitable vector or deliverysystem.

Suitable vectors and delivery systems include viral, such as systemsbased on adenovirus, vaccinia virus, retroviruses, herpes virus,adeno-associated virus or hybrids containing elements of more than onevirus. Non-viral delivery systems include cationic lipids and cationicpolymers as are well known in the art of DNA delivery. Physicaldelivery, such as via a “gene-gun” may also be used. The peptide orpeptide encoded by the nucleic acid may be a fusion protein, for examplewith an epitope which stimulates CD4-positive T-cells.

The peptide for use in a cancer vaccine may be any suitable peptide. Inparticular, it may be a suitable 9-mer peptide or a suitable 7-mer or8-mer or 10-mer or 11-mer peptide or 12-mer. Longer peptides may also besuitable, but 9-mer or 10-mer peptides as described in the attachedTable 1 are preferred.

Suitably, any nucleic acid administered to the patient is sterile andpyrogen free. Naked DNA may be given intramuscularly or intradermally orsubcutaneously. The peptides may be given intramuscularly,intradermally, intraperitoneally, intravenously or subcutaneously (seealso above regarding the method of producing a peptide). Preferably, thepeptides as active pharmaceutical components are given in combinationwith an adjuvant, such as, for example, IL-2, IL-12, GM-CSF, incompleteFreund's adjuvant, complete Freund's adjuvant or liposomal formulations.The most preferred adjuvants can be found in, for example, Brinkman J A,Fausch S C, Weber J S, Kast W M. Peptide-based vaccines for cancerimmunotherapy. Expert Opin Biol Ther. 2004 February; 4(2):181-98.

Vaccination results in CTL responses stimulated by professional antigenpresenting cells; once CTL are primed, there may be an advantage inenhancing MHC expression in tumor cells.

It may also be useful to target the vaccine to specific cellpopulations, for example antigen presenting cells, either by the site ofinjection, use of targeting vectors and delivery systems, or selectivepurification of such a cell population from the patient and ex vivoadministration of the peptide or nucleic acid (for example dendriticcells may be sorted as described in Zhou et al. (1995) Blood 86,3295-3301; Roth et al. (1996) Scand. J. Immunology 43, 646-651). Forexample, targeting vectors may comprise a tissue- or tumour-specificpromoter which directs expression of the antigen at a suitable place.

A further aspect of the invention therefore provides a vaccine effectiveagainst cancer, or cancer or tumour cells, comprising an effectiveamount of a peptide according to the invention, or comprising a nucleicacid encoding such a peptide. It is also preferred that the vaccine is anucleic acid vaccine. It is known that inoculation with a nucleic acidvaccine, such as a DNA vaccine, encoding a polypeptide leads to a T cellresponse. Most preferred is a vaccine comprising a (synthetic) peptideor peptides (i.e. either alone or in combinations of 1, 2, 3, 4, 5 or 6,11 or even more peptides, see also further below).

Conveniently, the nucleic acid vaccine may comprise any suitable nucleicacid delivery means. The nucleic acid, preferably DNA, may be naked(i.e. with substantially no other components to be administered) or itmay be delivered in a liposome or as part of a viral vector deliverysystem.

It is believed that uptake of the nucleic acid and expression of theencoded polypeptide by dendritic cells may be the mechanism of primingof the immune response; however, dendritic cells may not be transfectedbut are still important since they may pick up expressed peptide fromtransfected cells in the tissue.

Preferably the vaccine, such as DNA vaccine, is administered into themuscle or into the skin. The nucleic acid vaccine may be administeredwithout adjuvant. The nucleic acid vaccine may also be administered withan adjuvant such as BCG or alum. Other suitable adjuvants includeAquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and proprietory adjuvants such as Ribi's Detox. QuilA, another saponin derived adjuvant, may also be used (Superfos,Denmark). Other adjuvants such as Freund's may also be useful. Thenucleic acid vaccine is administered without adjuvant. It may also beuseful to give the peptide conjugated to keyhole limpet haemocyanin,preferably also with an adjuvant.

Polynucleotide-mediated immunisation therapy of cancer is described inConry et al. (1996) Seminars in Oncology 23, 135-147; Condon et al(1996) Nature Medicine 2, 1122-1127; Gong et al. (1997) Nature Medicine3, 558-561; Zhai et al. (1996) J. Immunol. 156, 700-710; Graham et al.(1996) Int J. Cancer 65, 664-670; and Burchell et al. (1996) pp 309-313In: Breast Cancer, Advances in biology and therapeutics, Calvo et al.(eds), John Libbey Eurotext, all of which are incorporated herein byreference in their entireties.

A still further aspect of the present invention provides the use of apeptide according to the invention, or of a polynucleotide or expressionvector encoding such a peptide, in the manufacture of a medicament forkilling target cells in a patient that target cells aberrantly express apolypeptide comprising an amino acid sequence of the invention.

A still further aspect of the present invention provides the use of apeptide according to the invention, or of a polynucleotide or expressionvector encoding such a peptide, for the manufacture of a medicament forinducing an immune response, in particular a cellular immune response,more particularly a T-cell mediated immune response against cells ofsolid tumours which cells express a human class II MHC molecule on theirsurface and present a polypeptide comprising an amino acid sequence ofthe invention. It has been surprisingly found in the context of thepresent invention that tumour cells of solid tumours, in contrast tohealthy cells of the same tissue, express human HLA class II molecule ontheir surface.

A further aspect of the invention thus provides a method for producingactivated cytotoxic T lymphocytes (CTL) in vivo or in vitro, the methodcomprising contacting in vitro CTL with antigen-loaded human class IIMHC molecules expressed on the surface of a suitable antigen-presentingcell for a period of time sufficient to activate, in an antigen specificmanner, said CTL wherein the antigen is a peptide according to theinvention.

Suitably, the CTL are CD4-positive helper cells, preferably of TH1-type.The MHC class II molecules may be expressed on the surface of anysuitable cell and it is preferred if the cell is one which does notnaturally express MHC class II molecules (in which case the cell istransfected to express such a molecule) or, if it does, it is defectivein the antigen-processing or antigen-presenting pathways. In this way,it is possible for the cell expressing the MHC class II molecule to beprimed substantially completely with a chosen peptide antigen beforeactivating the CTL.

The antigen-presenting cell (or stimulator cell) typically has an MHCclass II molecule on its surface and preferably is substantiallyincapable of itself loading said MHC class II molecule with the selectedantigen. As is described in more detail below, the MHC class II moleculemay readily be loaded with the selected antigen in vitro.

Preferably the mammalian cell lacks or has a reduced level or hasreduced function of the TAP peptide transporter. Suitable cells thatlack the TAP peptide transporter include T2, RMA-S and Drosophila cells.TAP is the Transporter Associated with antigen Processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre and Ljunggren(1985) J. Exp. Med. 162, 1745.

It is preferable that the host cell expresses substantially no MHC classI molecules before transfection. Preferably, the stimulator cellexpresses a molecule important for T-cell costimulation, such as any ofB7.1, B7.2, ICAM-1 and LFA 3.

The nucleic acid sequences of numerous MHC class II molecules, and ofthe costimulator molecules, are publicly available from the GenBank andEMBL databases.

In a further embodiment, combinations of HLA molecules may also be used,such as, for example, MHC-class II molecules as described in the TablesA and B herein. The use of recombinant polyepitope vaccines for thedelivery of multiple CD8⁺ CTL epitopes is described in Thomson et al.(1996) J. Immunol. 157, 822-826 and WO 96/03144, both of which areincorporated herein by reference. In relation to the present invention,it may be desirable to include in a single vaccine, a peptide (or anucleic acid encoding a peptide) wherein the peptide includes, in anyorder, an amino acid sequence of the present invention and another CD8⁺T cell-stimulating epitope. Such a vaccine would be particularly usefulfor treating cancers. Such “bead-on-a-string” vaccines are typically DNAvaccines. The simultaneous triggering of an MHC class II-dependentimmune response together with an MHC class I-dependent immune responsehas the advantage that this leads to a local TH₁-like T-cell-reaction ofCD4-positive T-cells, whereby the MHC class I-dependent CD8-positiveT-cells are supported.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al. (1995) Proc. Natl.Acad. Sci. USA 92, 432-436 and Kawakami et al. (1992) J. Immunol. 148,638643 use autologous tumor-infiltrating lymphocytes in the generationof CTL. Plebanski et al. (1995) Eur. J. Immunol. 25, 1783-1787 makes useof autologous peripheral blood lymphocytes (PLBs) in the preparation ofCTL. Jochmus et al. (1997) J. Gen. Virol. 78, 1689-1695 describes theproduction of autologous CTL by employing pulsing dendritic cells withpeptide or polypeptide, or via infection with recombinant virus. Hill etal. (1995) J. Exp. Med. 181, 2221-2228 and Jerome et al. (1993) J.Immunol. 151, 1654-1662 make use of B cells in the production ofautologous CTL. In addition, macrophages pulsed with peptide orpolypeptide, or infected with recombinant virus, may be used in thepreparation of autologous CTL. S. Walter et al. Cutting edge:predetermined avidity of human CD8 T cells expanded on calibratedMHC/anti-CD28-coated microspheres. J. Immunol. 2003 Nov. 15;171(10):4974-8 describe the in vitro priming of T cells by usingartificial antigen presenting cells, which is also a suitable way forgenerating T cells against the peptide of choice.

Allogeneic cells may also be used in the preparation of CTL and thismethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insects cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (1994) Virology 202, 449-955), which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated CTL that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated CTL obtainable by the foregoing methods of theinvention.

A still further aspect of the invention provides activated CTL thatselectively recognise a cell that aberrantly expresses a polypeptidecomprising an amino acid sequence of the invention. Preferably, the CTLrecognises the cell by interacting with the HLA/peptide-complex (forexample, binding). The CTL are useful in a method of killing targetcells in a patient, the method comprising targeting cells thataberrantly express a polypeptide comprising an amino acid sequence ofthe invention wherein the patient is administered an effective number ofthe activated CTL. The CTL that are administered to the patient may bederived from the patient and activated as described above (i.e. they areautologous CTL). Alternatively, the CTL are not from the patient but arefrom another individual. Of course, it is preferred if the individual isa healthy individual. By “healthy individual” the inventors mean thatthe individual is generally in good health, preferably has a competentimmune system and, more preferably, is not suffering from any diseasewhich can be readily tested for, and detected.

The activated CTL express a T-cell receptor (TCR), that is involved inrecognising cells that express the aberrant polypeptide. It is useful ifthe cDNA encoding the TCR is cloned from the activated CTL andtransferred into a further CTL for expression.

In vivo, the target cells for the CD4-positive CTL according to thepresent invention can be cells of the tumour (which sometimes expressMHC class II) and/or stromal cells surrounding the tumour (tumour cells)(which sometimes also express MHC class II).

The TCRs of CTL clones of the invention specific for the peptides of theinvention are cloned. The TCR usage in the CTL clones is determinedusing (i) TCR variable region-specific monoclonal antibodies and (ii) RTPCR with primers specific for Va and Vp gene families. A cDNA library isprepared from poly-A mRNA extracted from the CTL clones. Primersspecific for the C-terminal portion of the TCR a and P chains and forthe N-terminal portion of the identified Va and P segments are used. Thecomplete cDNA for the TCR a chain is amplified with a high fidelity DNApolymerase and the amplified products cloned into a suitable cloningvector. The cloned a and P chain genes may be assembled into a singlechain TCR by the method as described by Chung et al. (1994) Proc. Natl.Acad. Sci. USA 91, 12654-12658. In this single chain construct, the VaJsegment is followed by the V DJ segment, followed by the Cp segmentfollowed by the transmembrane and cytoplasmic segment of the CD3 chain.This single chain TCR is then inserted into a retroviral expressionvector (a panel of vectors may be used based on their ability to infectmature human CD8-positive T lymphocytes and to mediate gene expression:the retroviral vector system Kat is one preferred possibility (see Fineret al. (1994) Blood 83, 43). High titre amphotrophic retrovirus are usedto infect purified CD8-positive or CD4-positive T lymphocytes isolatedfrom the peripheral blood of tumour patients (following a protocolpublished by Roberts et al. (1994) Blood 84, 2878-2889, incorporatedherein by reference). Anti-CD3 antibodies are used to triggerproliferation of purified CD8⁺ T-cells, which facilitates retroviralintegration and stable expression of single chain TCRs. The efficiencyof retroviral transduction is determined by staining of infected CD8⁺T-cells with antibodies specific for the single chain TCR. In vitroanalysis of transduced CD8-positive T-cells establishes that theydisplay the same tumour-specific killing as seen with theallo-restricted CTL clone from which the TCR chains were originallycloned. Populations of transduced CD8-positive T-cells with the expectedspecificity may be used for adoptive immunotherapy of the tumourpatients. Patients may be treated with in between 10⁸ to 10¹¹autologous, transduced CTL. Analogously to CD8-positive, transducedCD4-positive T helper cells carrying related constructs can begenerated.

Other suitable systems for introducing genes into CTL are described inMoritz et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4318-4322,incorporated herein by reference. Eshhar et al (1993) Proc. Natl. Acad.Sci. USA 90, 720-724 and Hwu et al (1993) J. Exp. Med. 178, 361-366 alsodescribe the transfection of CTL. Thus, a further aspect of theinvention provides a TCR that recognises a cell that aberrantlyexpresses a polypeptide comprising an amino acid sequence of theinvention, the TCR being obtainable from the activated CTL.

In addition to the TCR, functionally equivalent molecules to the TCR areincluded in the invention. These include any molecule which isfunctionally equivalent to a TCR that can perform the same function as aTCR. In particular, such molecules include genetically engineeredthree-domain single-chain TCRs as made by the method described by Chunget al. (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658, incorporatedherein by reference, and referred to above. The invention also includesa polynucleotide encoding the TCR or functionally equivalent molecule,and an expression vector encoding the TCR or functionally equivalentmolecule thereof. Expression vectors that are suitable for expressingthe TCR of the invention include those described above in respect ofexpression of the peptides of the invention.

It is, however, preferred that the expression vectors are ones that areable to express the TCR in a CTL following transfection.

A still further aspect of the invention provides a method of killingtarget cells in a patient that target cells aberrantly expressing apolypeptide comprising an amino acid sequence of the invention, themethod comprising the steps of (1) obtaining CTL from the patient; (2)introducing into said cells a polynucleotide encoding a TCR, or afunctionally equivalent molecule, as defined above; and (3) introducingthe cells produced in step (2) into the patient.

A still further aspect of the invention provides a method of killingtarget cells in a patient that target cells aberrantly expressing apolypeptide comprising a peptide of the present invention as discussedabove the method comprising the steps of (1) obtaining antigenpresenting cells, such as dendritic cells, from said patient; (2)contacting said antigen presenting cells with a peptide of the presentinvention, or with a polynucleotide encoding such a peptide, ex vivo;and (3) reintroducing the so treated antigen presenting cells into thepatient.

Preferably, the antigen presenting cells are dendritic cells. Suitably,the dendritic cells are autologous dendritic cells that are pulsed withan antigenic peptide. The antigenic peptide may be any suitableantigenic peptide that gives rise to an appropriate T-cell response.T-cell therapy using autologous dendritic cells pulsed with peptidesfrom a tumour associated antigen is disclosed in Murphy et al. (1996)The Prostate 29, 371-380 and Tjua et al (1997) The Prostate 32, 272-278.

In a further embodiment, the antigen presenting cells, such as dendriticcells, are contacted with a polynucleotide that encodes a peptide of theinvention. The polynucleotide may be any suitable polynucleotide and ispreferably capable of transducing the dendritic cell thereby resultingin the presentation of a peptide and induction of immunity.

Preferably, the polynucleotide may be comprised in a viralpolynucleotide or virus. For example, adenovirus-transduced dendriticcells have been shown to induce antigen-specific antitumour immunity inrelation to MUC1 (see Gong et al. (1997) Gene Ther. 4, 1023-1028).Similarly, adenovirus-based systems may be used (see, for example, Wanet al. (1997) Hum. Gene Ther. 8, 1355-1363); retroviral systems may beused (Specht et al. (1997) J. Exp. Med. 186, 1213-1221 and Szabolcs etal. (1997) Blood particle-mediated transfer to dendritic cells may alsobe used (Tuting et al. (1997) Eur. J. Immunol. 27, 2702-2707); and RNAmay also be used (Ashley et al. (1997) J. Exp. Med. 186, 1177 1182).

It will be appreciated that, with respect to the methods of killingtarget cells in a patient, it is particularly preferred that the targetcells are cancer cells, more preferably renal or colon cancer cells.

More particularly, preferably, the patients who are treated by themethods of the invention have the HLA-DR haplotype. Thus, in a preferredembodiment the HLA haplotype of the patient is determined prior totreatment. HLA haplotyping may be carried out using any suitable method.Such methods are well known in the art.

The invention includes the use of the peptides of the invention (orpolynucleotides encoding them) for active in vivo vaccination; formanipulation of autologous dendritic cells in vitro followed byintroduction of the so-manipulated dendritic cells in vivo to activateCTL responses; to activate autologous CTL in vitro followed by adoptivetherapy (i.e. the so-manipulated CTL are introduced into the patient);and to activate CTL from healthy donors (MHC matched or mismatched) invitro followed by adoptive therapy.

In a preferred embodiment, vaccines of the present invention areadministered to a host either alone or in combination with anothercancer therapy to inhibit or suppress the formation of tumours.

Peptide vaccines of the present invention may be administered withoutadjuvant. The peptide vaccine may also be administered with an adjuvantsuch as BCG or alum. Other suitable adjuvants include Aquila's QS21stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived fromsaponin, mycobacterial extracts and synthetic bacterial cell wallmimics, and proprietory adjuvants such as Ribi's Detox. Quil A, anothersaponin derived adjuvant, may also be used (Superfos, Denmark). Otheradjuvants such as CpG oligonucleotides, stabilized RNA, Imiquimod(commercially available under the tradename Aldara™ from 3M Pharma,U.S.A.), Incomplete Freund's Adjuvant (commercially available asMontanide ISA-51 from Seppic S. A., Paris, France), liposomalformulations or GM-CSF may also be useful. It may also be useful to givethe peptide conjugated to keyhole limpet hemocyanin, preferably alsowith an adjuvant.

Peptides according to the invention can also be used as diagnosticreagents. Using the peptides it can be analysed, whether in aCTL-population, CTLs are present that are specifically directed againsta peptide or are induced by a therapy. Furthermore, the increase ofprecursor T-cells can be tested with those peptides that have reactivityagainst the defined peptide. Furthermore, the peptide can be used asmarker in order to monitor the progression of the disease of a tumourthat expresses the antigen from which the peptide is derived.

In the attached Table 1 the peptides as identified are listed. Inaddition, in the Table the proteins are designated, from which thepeptide is derived, and the respective position of the peptide in therespective protein. Furthermore the respective Acc-Numbers are giventhat relate to the Genbank of the “National Centre for BiotechnologyInformation” of the National Institute of Health (seehttp:www.ncbi.nlm.nih.gov).

In another preferred embodiment peptides of the present invention areused for staining of leukocytes, in particular of T-lymphocytes. Thisuse is of particular advantage if it should be proven, whether in aCTL-population specific CTLs are present that are directed against apeptide. Furthermore the peptide can be used as marker for determiningthe progression of a therapy in a tumourous disease or disorder.

In another preferred embodiment peptides of the present invention areused for the production of an antibody. Polyclonal antibodies can beobtained in a standard fashion by immunisation of animals via injectionof the peptide and subsequent purification of the immune globulin.Monoclonal antibodies can be produced according to standard protocolssuch as described, for example, in Methods Enzymol. (1986), 121,Hybridoma technology and monoclonal antibodies.

The invention also provides a pharmaceutical composition that containsone or more peptides according to the invention. This composition isused for parenteral administration, such as subcutaneous, intradermal,intramuscular or oral administration. For this, the peptides aredissolved or suspended in a pharmaceutically acceptable, preferablyaqueous carrier. In addition, the composition can contain excipients,such as buffers, binding agents, blasting agents, diluents, flavours,lubricants, etc. The peptides can also be administered together withimmune stimulating substances, such as cytokines. An extensive listingof excipients that can be used in such a composition, can be, forexample, taken from A. Kibbe, Handbook of Pharmaceutical Excipients, 3.Ed., 2000, American Pharmaceutical Association and pharmaceutical press.The composition can be used for a prevention, prophylaxis and/or therapyof tumourous diseases.

The pharmaceutical preparation, containing at least one of the peptidesof the present invention comprising any of the SEQ ID No. 1 to SEQ IDNo. 49 is administered to a patient that suffers from a tumourousdisease that is associated with the respective peptide or antigen. Bythis, a CTL-specific immune response can be triggered.

In another aspect of the present invention, a combination of two orseveral peptides according to the present invention can be used asvaccine, either in direct combination or within the same treatmentregimen. Furthermore, combinations with other peptides, for example MHCclass II specific peptides can be used. The person of skill will be ableto select preferred combinations of immunogenic peptides by testing, forexample, the generation of T-cells in vitro as well as their efficiencyand overall presence, the proliferation, affinity and expansion ofcertain T-cells for certain peptides, and the functionality of theT-cells, e.g. by analyzing the production of IFN-γ (see also examplesbelow), IL-12 or Perforin. Usually, the most efficient peptides are thencombined as a vaccine for the purposes as described above.

A preferred vaccine will contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 different peptides, preferably 4, 5, 6 or 7 different peptides,and most preferably 6 different peptides.

Finally, the vaccine can be dependent from the specific type of cancerthat the patient to be treated is suffering from as well as the statusof the disease, earlier treatment regimens, the immune status of thepatient, and, of course, the HLA-haplotype of the patient.

It has been shown that the 80 N-terminal amino acids of Ii aresufficient to direct proteins into the class II processing pathway(Sanderson, S., Frauwirth, K. & Shastri, N. (1995) Proc. Natl. Acad.Sci. U.S. A 92, 7217-7221, Wang, R. F., Wang, X., Atwood, A. C.,Topalian, S. L. & Rosenberg, S. A. (1999) Science 284, 1351-1354).

The identification of T-helper cell epitopes of tumour associatedantigens remains an important task in anti-tumour immunotherapy. Herethe inventors report a generally applicable method and peptides thathave been derived from differential peptide analysis by MS to identifynaturally processed and presented MHC class II ligands of tumourassociated antigens. This approach combines for the first time atransfection step of APC with a vector encoding for a fusion proteinbetween the Ii chain and the Ag of interest, elution of the HLA-boundpeptides and MS identification of the Ag-derived peptides presented bythe transfectant by comparison to the non-transfected cells. Moreover,the inventors could validate the method by showing that T-cells inducedagainst the identified peptide specifically recognise transfectantsoverexpressing the cognate Ag. Although the identified peptides stillhave to be tested for their immunogenicity in vivo, our approach leadsto the exact characterisation of naturally processed MHC class IIligands. Thus, the inventors avoid testing either synthetic overlappingpeptides of tumour associated antigens, or a broad range of peptidesselected by epitope prediction, which is less accurate as compared toclass I epitope prediction. In contrast to laborious T-cell assays,which might lead to the identification of cryptic T-cell epitopes unableto induce T-cell activation in vivo (Anderton, S. M., Viner, N. J.,Matharu, P., Lowrey, P. A. & Wraith, D. C. (2002) Nat. Immunol. 3,175-181), the work can be focused on the few peptides that are found tobe presented. Moreover, using this method it is not necessary to producethe recombinant Ag or to possess Ag-expressing tumour cell lines inorder to prove that the peptides are naturally processed.

The inventors used the N-terminus of Ii to direct tumour associatedantigens into the class II processing compartment of EBV-transformed Bcells. In order to achieve this the inventors constructed a versatilevector to express any antigen as a fusion protein with Ii and whichhelped determine the expression level of the protein in transfectedcells by Western blot analysis. It has already been shown that theN-terminus of Ii is sufficient to target proteins into the class IIprocessing compartment. But until now this has only been described in amodel using ovalbumin (Sanderson, S., Frauwirth, K. & Shastri, N. (1995)Proc. Natl. Acad. Sci. U.S. A 92, 7217-7221), in order to identifyunknown Ag using fusion protein-encoding cDNA libraries (Wang, R. F.,Wang, X., Atwood, A. C., Topalian, S. L. & Rosenberg, S. A. (1999)Science 284, 1351-1354) or to confirm the specificity of known T-cellclones (Chaux, P., Vantomme, V., Stroobant, V., Thielemans, K.,Corthals, J., Luiten, R., Eggermont, A. M., Boon, T. & van der, B. P.(1999) J. Exp. Med. 189, 767-778). To the inventors' knowledge thismethod has never been used before to identify naturally presented MHCclass II bound peptides of known tumour associated antigens. Thedifferential analysis of class II ligands of transfected andnon-transfected cells by MALDI-MS and the further characterisation ofthe differentially expressed peptides by ESI-MS results in astraightforward method for identifying class II ligands of antigens ofinterest. Transfection of cells with keratin 18 fusion proteins provedthat the inventors' method is generally applicable for antigens ofinterest, again, the inventors were also able to describe anHLA-DR-presented peptide from a model transgene, keratin 18.

The identification of helper T-cell epitopes of TAA remains an importanttask in anti-tumor immunotherapy. Until now, different strategies forthe identification of class II peptides from TAA have been carried out,ranging from the incubation of APCs with the antigen of interest inorder to be taken up and processed (Chaux, P., V. Vantomme, V.Stroobant, K. Thielemans, J. Corthals, R. Luiten, A. M. Eggermont, T.Boon, and B. P. van der Bruggen. 1999. Identification of MAGE-3 epitopespresented by HLA-DR molecules to CD4(+) T lymphocytes. J. Exp. Med.189:767-778), to various transfection strategies with fusion proteins(Dengjel, J., P. Decker, O, Schoor, F. Altenberend, T. Weinschenk, H. G.Rammensee, and S. Stevanovic. 2004. Identification of a naturallyprocessed cyclin D1 T-helper epitope by a novel combination of HLA classII targeting and differential mass spectrometry. Eur. J. Immunol.34:3644-3651). All these methods are very time-consuming and it oftenremained unclear, if the identified HLA ligands are actually presentedin vivo by human tissue. The inventors could show for the first timethat it is possible to isolate HLA class II ligands directly fromdissected solid tumors, thus identifying the peptides that are presentedby tumors and surrounding tissue in vivo, which can hence be recognizedby T-cells bearing the appropriate T-cell receptor and whichsimultaneously express the co-stimulatory ligand CD4 on their cellsurface. Among the proteins functioning as a source for endogenouslyprocessed HLA class II ligands, several housekeeping and immuno-relevantproteins were identified. However, peptides from TAA could also bedetected, leading to a straightforward approach for the identificationof in vivo relevant class II ligands of TAA.

The inventors identified three ligands accounting for one core sequencefrom IGFBP3 and one ligand from MMP7. The inventors found these proteinsto be over-expressed in renal cell carcinomas, in addition, they havebeen described as tumor-associated (Miyamoto, S., K. Yano, S. Sugimoto,G. Ishii, T. Hasebe, Y. Endoh, K. Kodama, M. Goya, T. Chiba, and A.Ochiai. 2004. Matrix metalloproteinase-7 facilitates insulin-like growthfactor bioavailability through its proteinase activity on insulin-likegrowth factor binding protein 3. Cancer Res. 64:665-671; Sumi, T., T.Nakatani, H. Yoshida, Y. Hyun, T. Yasui, Y. Matsumoto, E. Nakagawa, K.Sugimura, H. Kawashima, and O. Ishiko. 2003. Expression of matrixmetalloproteinases 7 and 2 in human renal cell carcinoma. Oncol. Rep.10:567-570; Cheung, C. W., D. A. Vesey, D. L. Nicol, and D. W. Johnson.2004. The roles of IGF-I and IGFBP-3 in the regulation of proximaltubule, and renal cell carcinoma cell proliferation. Kidney Int.65:1272-1279). These peptides bound promiscuously to HLA class IImolecules and were able to activate CD4-positive T-cells from differenthealthy donors. Thus, the inventors' approach will be helpful in theidentification of new class II peptide candidates from TAA for use inclinical vaccination protocols.

The invention in a further aspect relates to a method of killing targetcells in a patient, wherein the target cells express a polypeptidecomprising an amino acid sequence as provided herein, the methodcomprising administering to the patient an effective amount of a peptideaccording to the present invention or a nucleic acid according to thepresent invention or an expression vector according to the presentinvention, wherein the amount of said peptide or amount of said nucleicacid or amount of said expression vector is effective to provoke ananti-target cell immune response in said patient.

The invention in a further aspect relates to a method of killing targetcells in a patient, wherein the target cells express a polypeptidecomprising an amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof cytotoxic T lymphocytes (CTL) as defined according to the presentinvention.

The invention in a further aspect relates to a method of killing targetcells in a patient, wherein the target cells express a polypeptidecomprising an amino acid sequence according to the present invention,the method comprising the steps of (1) obtaining cytotoxic T lymphocytes(CTL) from the patient; (2) introducing into said cells a nucleic acidencoding a T cell receptor (TCR), or a functionally equivalent molecule,as defined according to the present invention; and (3) introducing thecells produced in step (2) into the patient.

Preferably, the target cells are cancer cells. More preferably, thecancer is leukemia or lymphoma that expresses a polypeptide whichcomprises an amino acid sequence of the present invention.

It has been surprisingly found in the context of the present inventionthat tumour cells of solid tumours, in contrast to healthy cells of thesame tissue, express human HLA class II molecule on their surface. Thisfact has been described only once in Brasanac et al. (Brasanac D,Markovic-Lipkovski J, Hadzi-Djokic J, Muller G A, Muller C A.Immunohistochemical analysis of HLA class II antigens and tumorinfiltrating mononuclear cells in renal cell carcinoma: correlation withclinical and histopathological data. Neoplasma. 1999; 46(3): 173-8),where cryostat sections of 37 renal cell carcinomas (RCC)-1-clear celltype, 10 granular and 2 chromophobe—were studied with indirectimmunoperoxidase method applying monoclonal antibodies (MoAb) to HLA-DR,-DP and -DQ antigens for analysis of HLA class II antigens, andanti-CD14, -CD3, -CD4 and -CD8 MoAb for tumour infiltrating mononuclearcells (TIM). The number of positive cells was estimatedsemiquantitatively and results of immunohistochemical investigation werecorrelated with clinical (patient age and sex, tumour size and TNMstage) and histopathological (cytology, histology, grade)characteristics of RCC. All RCC expressed HLA-DR, 92%-DQ and 73%-DPantigens with level of expression in hierarchy-DR>-DQ>-DP, but nostatistically important correlation could be established with any of thehistopathological or clinical parameters analyzed. Monocytes were moreabundant than T lymphocytes and CD4+ than CD8+ T cells, whereas tumourswith T lymphocyte predominance and approximately equal number of CD4+and CD8+ T cells had greatest average diameter. Inadequate activation ofT lymphocytes by tumour cells (despite capability of antigenpresentation) could be the reason for association of parameters whichindicates more aggressive tumour behaviour with aberrant HLA class IIantigen expression on RCC.

It should be understood that the features of the invention as disclosedand described herein can be used not only in the respective combinationas indicated but also in a singular fashion without departing from theintended scope of the present invention.

The invention will now be described in more detail by reference to thefollowing Figures, the Sequence listing, and the Examples. The followingexamples are provided for illustrative purposes only and are notintended to limit the invention.

SEQ ID NO: 1 to SEQ ID NO: 49 show peptide sequences of T-cell epitopecontaining peptides that are presented by MHC class II according to thepresent invention.

SEQ ID NO: 50 to SEQ ID NO: 79 show peptide sequences of Table 3.

TABLE 1 Peptide sequences aligned according to the motif ofHLA-DRB1*0101. Peptides with scores greater than 19 were considered asDRB1*0101 binders Sequence Gene SYFPEITHI SEQ ID −3 −2 −1 1 2 3 4 5 6 78 9 +1 +2 +3 Symbol Acc. Nr. Position Score NO. 1.   S Q D D I K G I Q KL Y G K R S MMP7 NP_002414 247-262 35 SEQ ID NO: 33 2.       N K Q K P IT P E T A E K L A R D CDC42 NP_426359 132-148 26 SEQ ID NO: 2 3.       DD P S T I E K L A K N K Q K P CDC42 NP_426359 121-136 19 SEQ ID NO: 3 4.            N P L K I F P S K R I L R R H CDH3 NP_001784  91-105 27 SEQID NO: 4 5.           E T G W L L L N K P L D R CDH3 NP_001784 163-17519 SEQ ID NO: 5 6.           D N E L Q E M S N Q G S K CLU NP_00182280-92 24 SEQ ID NO: 6 7.         A A G L L S T Y R A F L S S H COL15A1NP_001846 1243-1257 24 SEQ ID NO: 7 8. A P S L R P K D Y E V D A T L K SL N N Q COL1A2 NP_000080 1125-1145 25 SEQ ID NO: 8 9.       G P V D E VR E L Q K A I G A V P CTSD NP_001900 303-319 26 SEQ ID NO: 9 10.          I N H V V S V A G W G I S D G CTSZ NP_001327 239-253 33 SEQ IDNO: 10 11.   V P D D R D F E P S L G P V C P F R DCN NP_001911 40-57 23SEQ ID NO: 11 12. L P Q S I V Y K Y M S I R S D K S V P S EFEMP1NP_004096 389-408 30 SEQ ID NO: 12 13.         I V H R Y M T I T S E R SV P A EFEMP2 NP_058634 343-358 30 SEQ ID NO: 13 14.           K N G F VV L K G R P C K EIF5A NP_001961 27-39 28 SEQ ID NO: 14 15.       I T G YI I K Y E K P G S P P FN1 NP_002017 1930-1944 23 SEQ ID NO: 15 16.          G A T Y N I I V E A L K D Q FN1 NP_002017 2134-2147 20 SEQ IDNO: 16 17.   L T G Y R V R V T P K E K T G P FN1 NP_002017 1749-1764 21SEQ ID NO: 17 18.   I P G H L N S Y T I K G L K P G FN1 NP_002017659-674 24 SEQ ID NO: 18 19.               N L R F L A T T P N S L FN1NP_997640 1908-1919 26 SEQ ID NO: 19 20.         S N T D L V P A P A V RI L T P E GDF15 NP_004855 76-92 25 SEQ ID NO: 20 21.             A E I LE L A G N A A R D N H2AFJ NP_808760 61-74 32 SEQ ID NO: 21 22.         VK E P V A V L K A N R V W G A L HEXB NP_000512 153-169 32 SEQ ID NO: 2223.           T A E I L E L A G N A A R D N K HIST3H2A NP_254280 60-7532 SEQ ID NO: 23 24.     H P L H S K I I I I K K G H A K IGFBP3NP_000589 166-181 25 SEQ ID NO: 24 25.     H S K I I I I K K G H A K D SQ IGFBP3 NP_000589 169-184 28 SEQ ID NO: 25 26.     R P K H T R I S E LK A E A V K K D IGFBP5 NP_000590 138-155 32 SEQ ID NO: 26 27.       G PE D N V V I I Y L S R A G N P E ISLR NP_005536 380-397 26 SEQ ID NO: 2728.         S R P V I N I Q K T I T V T P N ITGA6 NP_000201 464-479 32SEQ ID NO: 28 29.   L D L S F N Q I A R L P S G L P V LUM NP_002336189-205 30 SEQ ID NO: 29 30.         K L P S V E G L H A I V V S D RMAP2K1IP1 NP_068805 12-27 32 SEQ ID NO: 30 31.         D T S T L E M M HA P R C G MMP12 NP_002417 80-93 23 SEQ ID NO: 31 32.   D Q N T I E T M RK P R C G N P D MMP2 NP_004521  90-106 20 SEQ ID NO: 32 33.         N PG E Y R V T A H A E G Y T P S AEBP1 NP_001120 947-963 20 SEQ ID NO: 134.           L D F L K A V D T N R A S V G PLXDC2 NP_116201 69-83 29SEQ ID NO: 34 35.                 H G N Q I A T N G V V H V I D R POSTNNP_006466 213-228 23 SEQ ID NO: 35 36.             R A I E A L H G H E LR P G RBM14 NP_006319 50-63 32 SEQ ID NO: 36 37. D P G V L D R M M K K LD T N S D S100A11 NP_005611 56-72 25 SEQ ID NO: 37 38.         N E E E IR A N V A V V S G A P SDCBP NP_001007069 56-71 26 SEQ ID NO: 38 39.          P A I L S E A S A P I P H SDCBP NP_001007068 29-41 24 SEQ IDNO: 39 40.             K V I Q A Q T A F S A N P A SDCBP NP_00100707014-27 30 SEQ ID NO: 40 41.         N G A Y K A I P V A Q D L N A P SSPP1 NP_000573 185-201 19 SEQ ID NO: 41 42.           T N G V V H V I TN V L Q P P A TGFBI NP_000349 621-636 29 SEQ ID NO: 42 43.         T T TQ L Y T D R T E K L R P E TGFBI NP_000349 116-131 23 SEQ ID NO: 43 44.            G K K E Y L I A G K A E G D G TIMP2 NP_003246 106-120 25 SEQID NO: 44 45.                 M G E I A S F D K A K L K K T TMSB10NP_066926  6-20 20 SEQ ID NO: 45 46.           M A E I E K F D K S K L KK TMSB4Y NP_004193  6-19 19 SEQ ID NO: 46 47.           V V S S I E Q KT E G A E K K YWHAZ NP_003397 61-75 22 SEQ ID NO: 47 48.           H S KI I I I K K G H A K IGFBP3 NP_000589 169-181 25 SEQ ID NO: 48 49.              N P P S M V A A G S V V A A V CCND1 NP_444284 198-212 24SEQ ID NO: 49

EXAMPLES Material and Methods

MHC class II immunohistology: tumors were fixed in 4% phosphate-bufferedformaldehyde, embedded in paraffin, stained with hematoxylin-eosin andexamined by light microscopy. Diagnosis of the RCC was carried outaccording to routine histopathological and immunohistologicalinvestigations (Fleming, S, and M. O'Donnell. 2000. Surgical pathologyof renal epithelial neoplasms: recent advances and current status.Histopathology 36:195-202).

For immunohistological detection of MHC class II molecules or CD68molecules, respectively, 5 μm paraffin-embedded tissue sections werepretreated with 10 mM citrate buffer, pH 6, followed by incubationeither with a mouse anti-HLA-DR alpha-chain mAb (clone TAL.1B5, 1:50) orCD68 Ab (Clone PGM1, 1:50) (DAKO, Hamburg, Germany) or mouse IgG1 (2μg/ml, BD Biosciences Pharmingen, San Diego, USA) and visualized usingthe Ventana iView DAB detection kit (Nexes System, Ventana MedicalSystems, Illkirch, France). Tissue sections were counterstained withhematoxylin and finally embedded in Entellan.

Elution and molecular analysis of HLA-DR bound peptides: frozen tumorsamples were processed as previously described (Weinschenk, T., C.Gouttefangeas, M. Schirle, F. Obermayr, S. Walter, O, Schoor, R. Kurek,W. Loeser, K. H. Bichler, D. Wernet, S. Stevanovic, and H. G. Rammensee.2002. Integrated functional genomics approach for the design ofpatient-individual antitumor vaccines. Cancer Res. 62:5818-5827) andpeptides were isolated according to standard protocols (Dengjel, J., H.G. Rammensee, and S. Stevanovic. 2005. Glycan side chains on naturallypresented MHC class II ligands. J. Mass Spectrom. 40:100-104) using theHLA-DR specific mAb L243 (Lampson, L. A. and R. Levy. 1980. Twopopulations of Ia-like molecules on a human B cell line. J. Immunol.125:293-299).

Natural peptide mixtures were analyzed by a reversed phase Ultimate HPLCsystem (Dionex, Amsterdam, Netherlands) coupled to a Q-TOF I massspectrometer (Waters, Eschborn, Germany), or by a reversed phase CapLCHPLC system coupled to a Q-TOF Ultima API (Waters) as previouslydescribed (Lemmel, C., S. Weik, U. Eberle, J. Dengjel, T. Kratt, H. D.Becker, H. G. Rammensee, and S. Stevanovic. 2004. Differentialquantitative analysis of MHC ligands by mass spectrometry using stableisotope labeling. Nat. Biotechnol. 22:450-454). Fragment spectra wereanalyzed manually and automatically.

Gene expression analysis by high-density oligonucleotide microarrays:RNA isolation from tumor and autologous normal kidney specimens as wellas gene expression analysis by Affymetrix Human Genome U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA) wereperformed as described previously (Krüger, T., O, Schoor, C. Lemmel, B.Kraemer, C. Reichle, J. Dengjel, T. Weinschenk, M. Müller, J.Hennenlotter, A. Stenzl, H. G. Rammensee, and S. Stevanovic. 2004.Lessons to be learned from primary renal cell carcinomas: novel tumorantigens and HLA ligands for immunotherapy. Cancer Immunol. Immunother).Data were analyzed with the GCOS software (Affymetrix). Pairwisecomparisons between tumor and autologous normal kidney were calculatedusing the respective normal array as baseline. For RCC149 and RCC211 noautologous normal kidney array data were available. Therefore, pooledhealthy human kidney RNA was obtained commercially (Clontech,Heidelberg, Germany) and used as the baseline for these tumors.

Maturation of DCs: DCs were prepared using blood from healthy donors.Briefly, PBMCs were isolated using standard gradient centrifugation(Lymphocyte Separation Medium, PAA Laboratories GmbH, Pasching, Austria)and plated at a density of 7×10⁶ cells/ml in X-Vivo 15 medium. After 2hours at 37° C., non-adherent cells were removed and adherent monocytescultured for 6 days in X-Vivo medium with 100 ng/ml GM-CSF and 40 ng/mlIL-4 (AL-ImmunoTools, Friesoythe, Germany). On day 7, immature DCs wereactivated with 10 ng/ml TNF-α (R&D Systems, Wiesbaden, Germany) and 20μg/ml poly(IC) (Sigma Aldrich, Steinheim, Germany) for 3 days.

Generation of antigen-specific CD4-positive T-cells: 106 PBMCs per wellwere stimulated with 2×10⁵ peptide pulsed (5 μg/ml) autologous DCs.Cells were incubated in 96-well plates (7 wells per donor and perpeptide) with T-cell medium: supplemented RPMI 1640 in the presence of10 ng/ml IL-12 (Promocell, Heidelberg, Germany). After 3 to 4 days ofco-incubation at 37° C., fresh medium with 80 U/ml IL-2 (Proleukin,Chiron Corporation, Emeryville, Calif., USA) and 5 ng/ml IL-7(Promocell) was added. Restimulations were done with autologous PBMCsplus peptide every 6 to 8 days.

Intracellular IFNγ staining: After 3 and 4 rounds of stimulation, PBMCswere thawed, washed twice in X-Vivo 15 medium, resuspended at 10⁷cells/ml in T-cell medium and cultured overnight. On the next day,PBMCs, pulsed with 5 μg/ml peptide, were incubated with effector cellsin a ratio of 1:1 for 6 h. Golgi-Stop (Becton Dickinson, Heidelberg,Germany) was added for the final 4 h of incubation.

Cells were analyzed using a Cytofix/Cytopern Plus kit (Becton Dickinson)and CD4-FITC-(Immunotools), IFNγ-PE- and CD8-PerCP clone SK1-antibodies(Becton Dickinson). For negative controls, cells of seven wells werepooled and incubated either with irrelevant peptide or without peptide,respectively. Stimulation with PMA/Ionomycin was used for positivecontrol. Cells were analyzed on a three-color FACSCalibur (BectonDickinson).

Example 1 HLA Class II Expression by RCC

Under normal, non-inflammatory conditions, HLA class II molecules shouldonly be expressed by cells of the hematopoietic system and by the thymicepithelium (Mach, B., V. Steimle, E. Martinez-Soria, and W. Reith. 1996.Regulation of MHC class II genes: lessons from a disease. Annu. Rev.Immunol. 14:301-331). The situation changes during inflammation. HLAclass II expression can be induced in most cell types and tissues byIFNγ (Leib und Gut-Landmann, S., J. M. Waldburger, M. Krawczyk, L. A.Otten, T. Suter, A. Fontana, H. Acha-Orbea, and W. Reith. 2004.Mini-review: Specificity and expression of CIITA, the master regulatorof MHC class II genes. Eur. J. Immunol. 34:1513-1525). As RCC incidenceis often accompanied by inflammatory events (Blay, J. Y., J. F. Rossi,J. Wijdenes, C. Menetrier-Caux, S. Schemann, S, Negrier, T. Philip, andM. Favrot. 1997. Role of interleukin-6 in the paraneoplasticinflammatory syndrome associated with renal-cell carcinoma. Int. J.Cancer 72:424-430; Elsässer-Beile, U., M. Rindsfuser, T. Grussenmeyer,W. Schultze-Seemann, and U. Wetterauer. 2000. Enhanced expression ofIFN-gamma mRNA in CD4(+) or CD8(+) tumour-infiltrating lymphocytescompared to peripheral lymphocytes in patients with renal cell cancer.Br. J. Cancer 83:637-641), class II molecules are indeed expressed inthe vicinity of or by tumors, as has been reported.

Example 2 Immuno-histochemical Staining of HLA Class II Molecules

The inventors analyzed HLA class II expression of ten RCC specimenscomprising histological clear cell and papillary renal carcinoma byimmuno-histochemical staining and found that all investigated samplesrevealed class II positive tumor cells. As exemplified in FIG. 1A, apronounced HLA class II expression was often detected at the margin ofthe tumor. In these areas the inventors observed a close spatialcorrelation of HLA positive tumor cells with tumor infiltrating immunecells as illustrated by the visualization of CD68-positive macrophagesin a serial tissue section (FIG. 1B). In RCC revealing a more papillaryarchitecture, the expression of HLA class II molecules was more evenlydistributed throughout the tumor (FIG. 1C, E, G). The comparison of theHLA class II and CD68 immuno-histochemical staining patterns in serialtissue sections clearly demonstrates that in addition to macrophages,tumor cells also express HLA class II (FIGS. 1C,D and E,F). It has beenshown that IFNγ producing CD4-positive T_(HI) cells as well as NaturalKiller (NK) cells infiltrate RCC (Cozar, J. M., J. Canton, M. Tallada,A. Concha, T. Cabrera, F. Gamido, and O. F. Ruiz-Cabello. 2005. Analysisof NK cells and chemokine receptors in tumor infiltrating CD4 Tlymphocytes in human renal carcinomas. Cancer Immunol. Immunother). Asclass II positive tumor cells were found predominantly in outer parts ofdissected tumors, one could speculate that leukocytes attracted by thetumor produce IFNγ which acts on neighboring malignant cells. Theabnormal expression of HLA class II molecules in neoplastic tissue isnot restricted to RCC, it can also be detected in TCC and CCA. FIG. 4shows immuno-histochemical staining of sampled tissue from humanadenocarcinoma of the colon.

Example 3 Analysis of Expression of IFNγ and Gene Transcripts Induced byIFNγ

Additionally, the inventors investigated HLA class II expression bycomparative gene expression analysis using oligonucleotide microarrays.With this technique the inventors were able to assess the overall HLAclass II expression in the dissected tumors regardless of the expressingcell types. The inventors analyzed differential expression in fourtumors, RCC149, RCC180, RCC190, and RCC211, compared with normalreference kidney. In all four tumors genes encoding HLA class IImolecules were found to be over-expressed (Table 2). One possible reasonfor this might be an induced expression by IFNγ and for this reason theinventors looked for other genes known to be up-regulated by interferons(Kolchanov, N. A., E. V. Ignatieva, E. A. Ananko, O. A. Podkolodnaya, I.L. Stepanenko, T. I. Merkulova, M. A. Pozdnyakov, N. L. Podkolodny, A.N. Naumochkin, and A. G. Romashchenko. 2002. Transcription RegulatoryRegions Database (TRRD): its status in 2002. Nucleic Acids Res.30:312-317). Interestingly, a considerable number of such genes werefound to be over-expressed in one or more tumor samples. Table 2 showsinterferon-inducible genes which were up-regulated reproducibly in allfour samples, in accordance with the inventors' earlier findings(Weinschenk, T., C. Gouttefangeas, M. Schirle, F. Obermayr, S. Walter,O, Schoor, R. Kurek, W. Loeser, K. H. Bichler, D. Wemet, S. Stevanovic,and H. G. Rammensee. 2002. Integrated functional genomics approach forthe design of patient-individual antitumor vaccines. Cancer Res.62:5818-5827). Among them are LMP2, LMP7, and MECL1-proteins that areexchanged against constitutive subunits of the large proteolyticholoenzyme residing in the cytosol, the proteasome, to form theimmuno-proteasome. The exchange of normally expressed proteolyticsubunits of the proteasome against IFN-inducible subunits is a hallmarkprocess in an interferon-rich environment. Additionally, IFNγ wasdirectly assessed by quantitative real-time (RT) PCR (TaqMan). Thetumors displayed in Table 2 showed a 5- to 60-fold IFNγ mRNAover-expression compared with the autologous normal RNA samples from thesame donor (data not shown). Thus, the inventors' results indicate thatIFNγ might play an important role in RCC and be the reason for abundantclass II expression.

TABLE 2 mRNA expression of interferon-inducible genes. Expression intumour samples was compared with autologous normal kidney (RCC180,RCC190) or pooled healthy kidney (RCC149, RCC211). All genes showed an“increase” in the change-call algorithm of the GCOS software for allfour tumours and have been described as interferon-inducible. GeneEntrez -fold over-expression tumor vs. normal Symbol GeneID Gene TitleRCC149 RCC180 RCC190 RCC211 HLA-DPA1 3113 major histocompatibility 3.53.7 4.9 13.9 complex, class II, DP alpha 1 HLA-DPB1 3115 majorhistocompatibility 2.6 2.5 2.8 14.9 complex, class II, DP beta 1HLA-DQB1 3119 major histocompatibility 4.3 4.0 6.5 5.3 complex, classII, DQ beta 1 HLA-DRB1 3123 major histocompatibility 1.2 1.9 2.8 4.3complex, class II, DR beta 1 CXCL10 3627 chemokine (C—X—C motif) 1.1 3.210.6 24.3 ligand 10 FCGR1A 2209 Fc fragment of IgG, high 6.5 2.6 12.129.9 affinity Ia, receptor for (CD64) IFI16 3428 interferon, gamma- 8.63.0 4.3 11.3 inducible protein 16 IFI44 10561 interferon-induced protein2.8 1.4 2.5 2.8 44 OAS1 4938 2′,5′-oligoadenylate 3.5 2.3 2.6 5.3synthetase 1, 40/46 kDa PSMB8 5696 proteasome subunit, beta 2.6 4.3 6.16.5 type, 8 (LMP7) PSMB9 5698 proteasome subunit, beta 4.3 7.5 6.5 16.0type, 9 (LMP2) PSMB10 5699 proteasome subunit, beta 3.2 2.5 5.3 13.0type, 10 (MECL1) SP100 6672 nuclear antigen Sp100 4.0 1.1 1.5 2.8 TAP16890 transporter 1, ATP-binding 2.5 2.8 6.5 8.0 cassette, sub-family B(MDR/TAP) VCAM1 7412 vascular cell adhesion 5.7 5.3 3.2 12.1 molecule 1

Example 4 HLA-DR Ligands Isolated from Cancer Tissue

According to publicly available data, peptides bound by HLA class IImolecules expressed in solid tumor tissue have so far not been isolatedor identified by others. The inventors analyzed ten different RCC, threeCCA and one TCC samples, and were able to isolate HLA-DR ligands fromall samples, amounting to 453 peptides in total (data not shown).Peptide sequences were determined by coupling chromatographic separationand tandem-mass spectrometric analysis (LC-MS/MS), as previouslydescribed (Weinschenk, T., C. Gouttefangeas, M. Schirle, F. Obermayr, S.Walter, O, Schoor, R. Kurek, W. Loeser, K. H. Bichler, D. Wemet, S.Stevanovic, and H. G. Rammensee. 2002. Integrated functional genomicsapproach for the design of patient-individual antitumor vaccines. CancerRes. 62:5818-582; Schirle M, Keilholz W, Weber B, Gouttefangeas C,Dumrese T, Becker H D, Stevanovic S, Rammensee H G. Identification oftumor-associated MHC class I ligands by a novel T-cell-independentapproach. Eur J. Immunol. 2000; 30(8):2216-25). An example for the denovo sequencing of peptides by LC-MS/MS is given in FIGS. 5 a and 5 b.The deduced primary amino acid sequences of collision fragmentsannotated in FIGS. 5 a and 5 b is included in Table 5. The tumorspecimens differed in their HLA genotypes, in weight and in the totalnumber of identified HLA ligands. Table 3 shows a representative list ofpeptides and corresponding source proteins identified from one exemplarytumor sample, RCC190. Peptides were isolated from HLA class II moleculesof cells as was previously described (Dengjel, J., P. Decker, O, Schoor,F. Altenberend, T. Weinschenk, H. G. Rammensee, and S. Stevanovic. 2004.Identification of a naturally processed cyclin D1 T-helper epitope by anovel combination of HLA class II targeting and differential massspectrometry. Eur. J. Immunol. 34:3644-3651).

TABLE 3 Example list of HLA-DR ligands isolated from RCC190. Shown arethe core sequences of HLA-DR ligands isolated from RCC190 (HLA-DRB1*11,DRB1*15, DRB3, DRB5). Gene Entrez Peptide Sequence Symbol GeneID (SEQ IDNO:) Gene Title ACTG1 71 WISKQEYDESGPSIVHRKCF actin, gam- (SEQ ID NO:50) ma 1 pro- peptide ALB 213 LKKYLYEIARRHP albumin (SEQ ID NO: 51)precursor ALB 213 TLVEVSRNLGKVG albumin (SEQ ID NO: 52) precursor ALB213 TPTLVEVSRNLGKVGS albumin (SEQ ID NO: 53) precursor APOA2 336EKSKEQLTPLIKKAGTELVNF apolipopro- (SEQ ID NO: 54) tein A-II precursorAPOB 338 YPKSLHMYANRLLDHR apolipopro- (SEQ ID NO: 55) tein B precursorC1R 715 EPYYKMQTRAGSRE complement (SEQ ID NO: 56) component 1, r sub-component C4B 721 APPSGGPGFLSIERPDSRPP complement (SEQ ID NO: 57)component 4B propro- tein C4BPA 722 FGPIYNYKDTIVFK complement (SEQ IDNO: 58) component 4 binding protein, alpha CALR 811 SPDPSIYAYDNFCalreticu- (SEQ ID NO: 59) lin precur- sor CALR 811EPPVIQNPEYKGEWKPRQIDNPD Calreticu- (SEQ ID NO: 60) lin precur- sor CFL11072 GVIKVFNDMKVRK cofilin 1 (SEQ ID NO: 61) (non- muscle) CPE 1363APGYLAITKKVAVPY carboxypep- (SEQ ID NO: 62) tidase E precursor FCGBP8857 ASVDLKNTGREEFLTA Fc fragment (SEQ ID NO: 63) of IgG binding proteinFCN1 2219 GNHQFAKYKSFKVADE ficolin 1 (SEQ ID NO: 64) precursor FTL 2512VSHFFRELAEEKREG ferritin, (SEQ ID NO: 65) light poly- peptide FTL 2512TPDAMKAAMALEKK ferritin, (SEQ ID NO: 66) light poly- peptide GAPD 2597FVMGVNHEKYDN glyceralde- (SEQ ID NO: 67) hyde-3- phosphate dehydro-genase GAPD 2597 TGVFTTMEKAGAH glyceralde- (SEQ ID NO: 68) hyde-3-phosphate dehydro- genase GAPD 2597 ISWYDNEFGYSNRVVDLMAHMAS glyceralde-KE hyde-3- (SEQ ID NO: 69) phosphate dehydro- genase HIST1H1C 3006GTGASGSFKLNKKAASGEAKPK H1 histone (SEQ ID NO: 70) family, member 2HLA-DQB1 3119 DVGVYRAVTPQGRPD major his- (SEQ ID NO: 71) tocompati-bility com- plex, class II, DQ beta 1 precursor HLA-DRB1 3123DVGEFRAVTELGRPD major his- (SEQ ID NO: 72) tocompati- bility com- plex,class II, DR beta 1 precursor IGFBP3 3486 HPLHSKIIIIKKGHAK insulin- (SEQID NO: 73) like growth factor binding protein 3 KNG1 3827 DKDLFKAVDAALKKkininogen 1 (SEQ ID NO: 74) NPC2 10577 KDKTYSYLNKLPVK Niemann- (SEQ IDNO: 75) Pick di- sease, type C2 precur- sor S100A8 6279VIKMGVAAHKKSHEESHKE S100 cal- (SEQ ID NO: 76) cium-bind- ing protein A8SERPINA1 5265 MIEQNTKSPLFMGKVVNPTQK serine (or (SEQ ID NO: 77) cysteine)proteinase inhibitor, clade A (alpha-1 antipro- teinase, antitryp- sin),mem- ber 1 SOD1 6647 GPHFNPLSRKHGGPK superoxide (SEQ ID NO: 78)dismutase 1, soluble TF 7018 DPQTFYYAVAVVKKDS transferrin (SEQ ID NO:79)

There was no correlation between tumor weight and number of identifiedHLA ligands. Peptide source proteins could be divided into two groups.On the one hand, ligands which should be presented by leukocytes werefound, such as peptides from complement components, e.g., C3, C4A, C4binding protein alpha, and other proteins linked to specific functionsof cells of the immune system, e.g., CD14, and Fc fragment of IgGbinding protein. On the other hand, the inventors were able to disclosethe nature and characteristics of previously unknown peptides presentedby tumor cells from over-expressed TAA, for example from vimentin,matrix metalloproteinase 7, eukaryotic translation elongation factor 1alpha 1, and nicotinamide N-methyltransferase. This observation is inaccordance with immuno-histochemistry data (FIGS. 1 and 4) anddemonstrates that HLA class II positive tumor cells and infiltratingleukocytes were present in analyzed specimens and that the elutedpeptides stem from antigens found to be over-expressed in these distinctcell types.

To identify peptides from TAA, the inventors compared the sourceproteins for the individual ligands with over-expressed genes detectedby micro-array analysis of tumors (Weinschenk, T., C. Gouttefangeas, M.Schirle, F. Obermayr, S. Walter, O, Schoor, R. Kurek, W. Loeser, K. H.Bichler, D. Wemet, S. Stevanovic, and H. G. Rammensee. 2002. Integratedfunctional genomics approach for the design of patient-individualantitumor vaccines. Cancer Res. 62:5818-5827; Krüger, T., O, Schoor, C.Lemmel, B. Kraemer, C. Reichle, J. Dengjel, T. Weinschenk, M. Müller, J.Hennenlotter, A. Stenzl, H. G. Rammensee, and S. Stevanovic. 2004.Lessons to be learned from primary renal cell carcinomas: novel tumorantigens and HLA ligands for immunotherapy. Cancer Immunol. Immunother).The inventors identified a peptide from insulin-like growth factorbinding protein 3, IGFBP3₁₆₆₋₁₈₁ on RCC190. In addition, two variants ofthis peptide, IGFBP3₁₆₉₋₁₈₁ and IGFBP3₁₆₉₋₁₈₄, which contain the samesequence core motif that is both necessary and sufficient to allowbinding to HLA-DRB1*0101 (for reference, see Table 1), were found onTCC108. From the same tumor, a peptide from matrix metalloproteinase 7,MMP7₂₄₇₋₂₆₂, could be isolated (Table 1). At the mRNA level, MMP7 wasover-expressed in 13 and IGFBP3 in 22 of 23 analyzed RCC (data notshown). In total, out of 453 peptide sequences initially identified (notshown), the underlying antigens for 49 peptides (SEQ ID NOS: 1-49) havebeen identified to be tumor-associated, either by the experiments of theinventors (data included in this document), or by others (Miyamoto, S.,K. Yano, S. Sugimoto, G. Ishii, T. Hasebe, Y. Endoh, K. Kodama, M. Goya,T. Chiba, and A. Ochiai. 2004. Matrix metalloproteinase-7 facilitatesinsulin-like growth factor bioavailability through its proteinaseactivity on insulin-like growth factor binding protein 3. Cancer Res.64:665-671; Sumi, T., T. Nakatani, H. Yoshida, Y. Hyun, T. Yasui, Y.Matsumoto, E. Nakagawa, K. Sugimura, H. Kawashima, and O. Ishiko. 2003.Expression of matrix metalloproteinases 7 and 2 in human renal cellcarcinoma. Oncol. Rep. 10:567-570; Cheung, C. W., D. A. Vesey, D. L.Nicol, and D. W. Johnson. 2004. The roles of IGF-I and IGFBP-3 in theregulation of proximal tubule, and renal cell carcinoma cellproliferation. Kidney Int. 65:1272-1279; Hao, X., B. Sun, L. Hu, H.Lahdesmaki, V. Dunmire, Y. Feng, S. W. Zhang, H. Wang, C. Wu, H. Wang,G. N. Fuller, W. F. Symmans, I. Shmulevich, and W. Zhang. 2004.Differential gene and protein expression in primary breast malignanciesand their lymph node metastases as revealed by combined cDNA microarrayand tissue microarray analysis. Cancer 100:1110-1122; Helmke, B. M., M.Polychronidis, A. Benner, M. Thome, J. Arribas, and M. Deichmann. 2004.Melanoma metastasis is associated with enhanced expression of thesyntenin gene. Oncol. Rep. 12:221-228; Hofmann, H. S., G. Hansen, G.Richter, C. Taege, A. Simm, R. E. Silber, and S. Burdach. 2005. Matrixmetalloproteinase-12 expression correlates with local recurrence andmetastatic disease in non-small cell lung cancer patients. Clin. CancerRes. 11: 1086-1092; Kamai, T., T. Yamanishi, H. Shirataki, K. Takagi, H.Asami, Y. Ito, and K. Yoshida. 2004. Overexpression of RhoA, Rac1, andCdc42 GTPases is associated with progression in testicular cancer. Clin.Cancer Res. 10:4799-4805; Koninger, J., N. A. Giese, F. F. di Mola, P.Berberat, T. Giese, I. Esposito, M. G. Bachem, M. W. Buchler, and H.Friess. 2004. Overexpressed decorin in pancreatic cancer: potentialtumor growth inhibition and attenuation of chemotherapeutic action.Clin. Cancer Res. 10:4776-4783; Mori, M., H. Shimada, Y. Gunji, H.Matsubara, H. Hayashi, Y. Nimura, M. Kato, M. Takiguchi, T. Ochiai, andN. Seki. 2004. S100A11 gene identified by in-house cDNA microarray as anaccurate predictor of lymph node metastases of gastric cancer. Oncol.Rep. 11: 1287-1293; Nagler, D. K., S. Kruger, A. Kellner, E. Ziomek, R.Menard, P. Buhtz, M. Krams, A. Roessner, and U. Kellner. 2004.Up-regulation of cathepsin X in prostate cancer and prostaticintraepithelial neoplasia. Prostate 60:109-119; Nanda, A., P.Buckhaults, S. Seaman, N. Agrawal, P. Boutin, S. Shankara, M. Nacht, B.Teicher, J. Stampfl, S. Singh, B. Vogelstein, K. W. Kinzler, and C. B.St. 2004. Identification of a binding partner for the endothelial cellsurface proteins TEM7 and TEM7R. Cancer Res. 64:8507-8511; Patel, I. S.,P. Madan, S. Getsios, M. A. Bertrand, and C. D. MacCalman. 2003.Cadherin switching in ovarian cancer progression. Int. J. Cancer106:172-177; Santelli, G., D. Califano, G. Chiappetta, M. T. Vento, P.C. Bartoli, F. Zullo, F. Trapasso, G. Viglietto, and A. Fusco. 1999.Thymosin beta-10 gene overexpression is a general event in humancarcinogenesis. Am. J. Pathol. 155:799-804; Schneider, D., J. Kleeff, P.O. Berberat, Z. Zhu, M. Korc, H. Friess, and M. W. Buchler. 2002.Induction and expression of betaig-h3 in pancreatic cancer cells.Biochim. Biophys. Acta 1588:1-6; Welsh, J. B., L. M. Sapinoso, S. G.Kern, D. A. Brown, T. Liu, A. R. Bauskin, R. L. Ward, N. J. Hawkins, D.I. Quinn, P. J. Russell, R. L. Sutherland, S. N. Breit, C. A. Moskaluk,H. F. Frierson, Jr., and G. M. Hampton. 2003. Large-scale delineation ofsecreted protein biomarkers overexpressed in cancer tissue and serum.Proc. Natl. Acad. Sci. U. S. A 100:3410-3415; Xie, D., J. S. Sham, W. F.Zeng, L. H. Che, M. Zhang, H. X. Wu, H. L. Lin, J. M. Wen, S. H. Lau, L.Hu, and X. Y. Guan. 2005. Oncogenic role of clusterin overexpression inmultistage colorectal tumorigenesis and progression. World J.Gastroenterol. 11:3285-3289). Exemplary analysis of immuno-stimulatorypotential of peptides binding to common HLA-DR alleles reveals theexistence of antigen-specific CD4-positive T-cells against IGFBP3₁₆₉₋₁₈₁and MMP7₂₄₇₋₂₆₂.

Promiscuous binding of exemplary SEQ ID NO: 1 to several HLA-DR allelescan be revealed by several independent methods: ligands of certainMHC/HLA molecules carry chemical related amino acids in certainpositions of their primary sequence, which permit the definition of apeptide motif for every MHC/HLA allele (Falk K, Rotzschke O, StevanovicS, Jung G, Rammensee H G. Allele-specific motifs revealed by sequencingof self-peptides eluted from MHC molecules. Nature. 1991;351(6324):290-6). SYFPEITHI uses motif matrices deduced from refinedmotifs exclusively based on natural ligand analysis by Edman degradationand tandem mass spectrometry. These matrices allow the prediction ofpeptides from a given protein sequence presented on MHC class I or classII molecules (Rotzschke O, Falk K, Stevanovic S, Jung G, Walden P,Rammensee H G. Exact prediction of a natural T-cell epitope. Eur J.Immunol. 1991; 21(11):2891-4).

Applying the principles of the predictions made by the SYFPEITHIalgorithm (Rammensee, H. G., J. Bachmann, and S. Stevanovic. 1997. MHCLigands and Peptide Motifs. Springer-Verlag, Heidelberg, Germany;Rammensee H, Bachmann J, Emmerich N P, Bachor O A, Stevanovic S.SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics.1999; 50(3-4):213-9) to the aforesaid exemplary peptide sequence (SEQ IDNO: 1), the binding of SEQ ID NO: 1 to several common HLA-DR alleles(see Table 7) was ranked. The algorithm has been successfully used topredict class I and class II epitopes from various antigens, e.g., fromthe human TAA TRP2 (prediction of an HLA class I ligand) (34) and SSX2(prediction of an HLA class II ligand) (Neumann F, Wagner C, StevanovicS, Kubuschok B, Schormann C, Mischo A, Ertan K, Schmidt W, PfreundschuhM. Identification of an HLA-DR-restricted peptide epitope with apromiscuous binding pattern derived from the cancer testis antigenHOM-MEL-40/SSX2. Int J. Cancer. 2004; 112(4):661-8). The threshold of ascore of 18 or higher for significant binding was defined based on theanalysis of binding scores for previously published promiscuouslybinding HLA-DR peptide ligands. Promiscuous binding is defined asbinding of a peptide with good binding strength as indicated by a scoreof 18 in the SYFPEITHI test or higher to two or more different commonHLA-DR alleles. The most common HLA DR alleles are depicted in Table 7.The loci of HLA-A and HLA-DR are in linkage disequilibrium yieldingcombinations of HLA-A2 and specific HLA-DRs that are favored incomparison to others. The HLA-DR genotypes of the source tumors wereanalyzed and confirmed to be HLA-DRB1*11 and DRB1*15 in both cases. Thepreferred anchor amino acid residues for the most common HLA class IIalleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, andDRB1*1501) are depicted in Table 4. For example, the HLA class II alleleDRB1*0301 preferentially binds peptides in its binding groove thatfeature specific amino acid residues in positions 1, 4, 6, and 9 fromthe N- to the C-terminal end of the core sequence of any given peptide.Specifically, DRB1*0301 shows good binding, if the core sequence of apeptide has a Glutamate residue (D) in position 4, as well as either L,I, F, M, or V in position 1, as well as K, R, E, Q, or N in position 6,as well as either Y, L, or F in position 9.

TABLE 4 Peptide motifs of common HLA-DR alleles. Depicted are anchoramino acids in one letter code at the corresponding binding pockets.Position HLA allel −3 −2 −1 1 2 3 4 5 6 7 8 9 +1 +2 +3 DRB1*0101 Y L A LV A G I L I S A I V T V F M C N A N P F M Q Y W DRB1*0301 L D K Y I R LF E F M Q V N DRB1*0401 F P N D D Y W S E E W I T H H I L Q K K L V H NN V A R Q Q M D R R E S S T T Y Y A A C C I I L L M M V V DRB1*0701 F DN V Y E S I W H T L I K Y L N F V Q R S T Y DRB1*1101 W L R A Y I K G FV H S M P A F Y DRB1*1501 L F I V Y L I I V M F

Results from in silico analysis based on the computer algorithms for theprediction of interaction between HLA molecules and peptide sequencesprovided through www.syfpeithi.de indicate that the peptide MMP7₂₄₇₋₂₆₂SEQ ID NO: 1 binds promiscuously to several HLA-DR alleles. According toresults from the predictive analysis, the peptide with SEQ ID NO: 1receives a high binding score for interaction with DRB1*1101, DRB1*1501,DRB1*0401, DRB1*0301, and DRB1*0101 (Table 7). The HLA-DR allelesanalyzed in this test for interaction with the peptide amino acidsequence/strength of binding cover at least 69.6% of the HLA-A2 positiveCaucasian population (Mori M, Beatty P G, Graves M, Boucher K M, MilfordE L. HLA gene and haplotype frequencies in the North Americanpopulation: the National Marrow Donor Program Donor Registry.Transplantation. 1997; 64(7):1017-27). As there is presently nofrequency data available for HLA-DR15, the allele was not taken intoconsideration for calculating the resulting coverage of the HLA-A2positive Caucasian population. Thus, it is very likely that with SEQ IDNO: 1, the coverage of the population is even higher than 69.6%, whichindicates that the peptide has an excellent perspective for serving as acandidate for the development of pharmaceutical preparations for themajority of cancer patients.

However, application of prediction algorithms leads only to conclusiveresults, if the results from in silico analyses are combined withexperimental confirmation for promiscuous binding, as was shown byothers before, who failed to demonstrate any immune responses triggeredby a peptide sequences predicted to be a good binder (Bohm C M, Hanski ML, Stefanovic S, Rammensee H G, Stein H, Taylor-Papadimitriou J, RieckenE O, Hanski C. Identification of HLA-A2-restricted epitopes of thetumor-associated antigen MUC2 recognized by human cytotoxic T-cells. IntJ. Cancer. 1998; 75(5):688-93). The prediction of artifacts like in theafore-mentioned case can principally not be ruled out, as the algorithmsused for prediction do not take into account that a peptide sequence isnot necessarily generated in an in vivo situation (inside living cells).Experimental confirmation can be obtained by collecting in vitro datafrom biological test, e.g., by demonstrating the presence or lack ofimmunogenicity of a peptide. Hence, for experimental confirmation ofpromiscuous binding of SEQ ID NO: 1 was obtained by collecting such invitro data. To test the peptides for their immuno-stimulatory capacityby in vitro T-cell priming experiments, the shortest variants (“coresequence”) of the IGFBP3 peptides, IGFBP3₁₆₉₋₁₈₁, and of the MMP7peptide, MMP7₂₄₇₋₂₆₂, were used.

To generate antigen-specific CD4-positive T-cells and to test thepeptides for promiscuous binding, PBMCs of 4 healthy donors withdifferent HLA-DR alleles (FIG. 2), one of them carrying DRB1*1101, werestimulated using peptide-pulsed autologous DCs. In addition, the peptideCCND1₁₉₈₋₂₁₂, a known T-cell epitope (Dengjel, J., P. Decker, O, Schoor,F. Altenberend, T. Weinschenk, H. G. Rammensee, and S. Stevanovic. 2004.Identification of a naturally processed cyclin D1 T-helper epitope by anovel combination of HLA class II targeting and differential massspectrometry. Eur. J. Immunol. 34:3644-3651), was used as positivecontrol. As a read-out system for the generation of antigen-specificCD4-positive T-cells, IFNγ levels were assessed by flow cytometry.T-cells were analyzed after the third and fourth weekly stimulation byintracellular IFNγ staining plus CD4-FITC and CD8-PerCP staining todetermine the percentage of IFNγ-producing cells in specific T-cellsubpopulations. In all experiments, stimulations with irrelevant peptideand without peptide were performed as negative controls. IFNγ responsewas considered as positive if the percentage of IFNγ producingCD4-positive T-cells was more than two-fold higher compared withnegative controls (Horton, H., N. Russell, E. Moore, I. Frank, R. Baydo,C. Havenar-Daughton, D. Lee, M. Deers, M. Hudgens, K. Weinhold, and M.J. Mc Elrath. 2004. Correlation between interferon-gamma secretion andcytotoxicity, in virus-specific memory T-cells. J. Infect. Dis.190:1692-1696).

In three of four donors the inventors were able to generate specificCD4-positive T-cells for both peptides (FIG. 2). T-cell responses couldnot be observed in donor 4 after any stimulation. In donor 1, 0.05% to0.1% (FIG. 3) IFNγ producing CD4-positive T-cells were detected in allseven stimulation attempts after the third stimulation with peptideIGFBP3₁₆₉₋₁₈₁. These T-cells could be expanded in most cases by anadditional round of stimulation to 0.09% to 0.13%. IFNγ-producingCD4-positive T-cells specific for the peptide IGFBP3₁₆₉₋₁₈₁ were alsoobserved in donor 2 and donor 3, with maximal frequencies of 0.05% and0.07% IFNγ producing CD4⁺ T-cells.

Donors 1, 2, and 3 also showed CD4⁺ T-cells reactive to peptideMMP7₂₄₇₋₂₆₂. The highest frequencies of IFNγ producing CD4⁺ T-cellsspecific for the MMP7 peptide were found in donors 1 and 2,respectively. Donors 1, 2, and 3 showed IFNγ responses to peptideCCND1₁₉₈₋₂₁₂, which had already been described as an MHC classII-restricted T-cell epitope (Dengjel, J., P. Decker, O. Schoor, F.Altenberend, T. Weinschenk, H. G. Rammensee, and S. Stevanovic. 2004.Identification of a naturally processed cyclin D1 T-helper epitope by anovel combination of HLA class II targeting and differential massspectrometry. Eur. J. Immunol. 34:3644-3651).

Thus, peptides from IGFBP3, MMP7, and CCND1 are promiscuous HLA class IIbinders that are able to elicit CD4-positive T-cell responses in threeout of four healthy donors carrying different HLA DR alleles. If the HLAalleles of the two tumour patients from which the IGFBP3 and MMP7peptides were derived are compared to those of the four healthy donors,it becomes obvious that the peptides are presented by HLA-DRB1*01,HLA-DRB1*04 and HLA-DRB1*11. All three aforesaid HLA DR allotypes have aglycine amino acid residue at position 86, and an aspartic acid residueat position 57 of their β chains, respectively (seewww.anthonynolan.com/HIG). Therefore, they have very similar bindingcharacteristics for their binding pockets P1 and P9 (Rammensee, H. G.,J. Bachmann, and S. Stevanovic. 1997. MHC Ligands and Peptide Motifs.Springer-Verlag, Heidelberg, Germany). For peptide CCND1₁₉₈₋₂₁₂, aT-cell epitope known to be presented by HLA-DRB1*0401 and HLA-DRB1*0408(Dengjel, J., P. Decker, O. Schoor, F. Altenberend, T. Weinschenk, H. G.Rammensee, and S. Stevanovic. 2004. Identification of a naturallyprocessed cyclin D1 T-helper epitope by a novel combination of HLA classII targeting and differential mass spectrometry. Eur. J. Immunol.34:3644-3651), the same holds true. Donor 4 carries HLA-DRB1*0318 andHLA-DRB1*1401, alleles with peptide motifs that differ in the primaryamino acid sequence of their beta chains from those described above.This could explain why it was not possible to elicit T-cell responsesagainst the two peptides using cells from this donor.

Interestingly, IFNγ-producing CD8-positive killer T-cells were detectedin two donors after stimulations with the three peptides, in particularin donor 3, but also to a lesser extent in donor 1 (data not shown).

TABLE 5 Mass Fragment [M + H]+ Amino acid sequence b2 216.1 SQ y4 447.3GKRS y6 723.4 LYGKRS y7 851.5 KLYGKRS y8 979.6 QKLYGKRS y9 1092.6IQKLYGKRS y10 1149.7 GIQKLYGKRS y11 1277.8 KGIQKLYGKRS y12 1390.8IKGIQKLYGKRS y13 1505.9 DIKGIQKLYGKRS y14 1620.9 DDIKGIQKLYGKRS R 129.1immonium R

TABLE 6 Haplotype frequencies of North American Caucasian population.Shown are the serological haplotypes. Haplotype HLA-A HLA-DR Frequency[%] 2 1 8.8 2 2 14.9 2 3 6.1 2 4 21.3 2 5 1.2 2 6 15.2 2 7 13.0 2 8 4.22 9 1.2 2 10  1.4 2 11  8.7 2 12  2.6 2 n.a. 1.4 “n.a.” stands forassigned.

TABLE 7 Binding scores of SEQ ID NO: 1 to common HLA-DR alleles Shownare the SEQ ID NO: 1 and SEQ ID NO: 25 SYFPEITHI binding scores for themost common HLA-DRB1 alleles in Caucasian populations. The frequenciesof the corresponding serological haplotypes of HLA-A2 positiveCaucasians are given in brackets. The peptides are considered to bindsufficiently well to a HLA class II molecule, if the score was equal to-or higher than 18. DRB1* allele 0101 0301 0401 0701 1101 1501 Antigen(8.8%) (6.1%) (21.3%) (13.0%) (8.7%) (n.a. %) SEQ ID NO: 1 35 18 20 1426 20 SEQ ID 28 28 20 18 26 18 NO: 25

1. An isolated tumour associated peptide comprising the peptide setforth in SEQ ID NO:2 flanked by N- and/or C-terminal extensions between1 to 10 amino acids in length.
 2. The isolated tumour associated peptideaccording to claim 1 wherein said isolated tumor associated peptide hasthe ability to bind human major histocompatibility complex (MHC)class-II molecule HLA-DRB*0101, and has the ability to bind to at leastone additional molecule of the human major histocompatibility complex(MHC) class-II.
 3. A fusion protein comprising the peptide set forth inSEQ ID NO: 2 flanked by N- and/or C-terminal extensions between 1 to 10amino acids in length and N-terminal amino acids of HLA-DRantigen-associated invariant chain (Ii).
 4. A composition comprising thetumour associated peptide of claim 1 and a pharmaceutically acceptablecarrier.
 5. A composition comprising the fusion protein of claim 3 and apharmaceutically acceptable carrier.